Epigenetic plasticity of hTERT gene promoter determines retinoid capacity to repress telomerase in maturation-resistant acute promyelocytic leukemia cells.

The expression of hTERT gene, encoding the catalytic subunit of telomerase, is a feature of most cancer cells. Changes in the chromatin environment of its promoter and binding of transcriptional factors have been reported in differentiating cells when its transcription is repressed. However, it is not clear whether these changes are directly involved in this repression or only linked to differentiation. In a maturation-resistant acute promyelocytic leukemia (APL) cell line (NB4-LR1), we have previously identified a new pathway of retinoid-induced hTERT repression independent of differentiation. Using a variant of this cell line (NB4-LR1(SFD)), which resists to this repression, we show that although distinct patterns of histone modifications and transcription factor binding at the proximal domain of hTERT gene promoter could concur to modulate its expression, this region is not sufficient to the on/off switch of hTERT by retinoids. DNA methylation analysis of the hTERT promoter led to the identification of two distinct functional domains, a proximal one, fully unmethylated in both cell lines, and a distal one, significantly methylated in NB4-LR1(SFD) cells, whose methylation was further re-enforced by retinoid treatment. Interestingly, we showed that the binding to this distal domain of a known hTERT repressor, WT1, was defective only in NB4-LR1(SFD) cells. We propose that epigenetic modifications targeting this distal region could modulate the binding of hTERT repressors and account either for hTERT reactivation and resistance to retinoid-induced hTERT downregulation.

Re-expression of DNA methylation-silenced CD44 gene in a resistant NB4 cell line: rescue of CD44-dependent cell death by cAMP.

In the acute promyelocytic leukemia cell line, NB4, activation of the CD44 receptor triggers apoptosis. This pathway does not operate in the retinoid-maturation-resistant NB4-LR1 subclone. In this work, we show that the CD44 gene is silenced in these cells. The molecular defect involves DNA methylation of cytosine phosphate guanine (CpG) island and underacetylation of histone H3 at CD44 promoter. The methylating inhibitor 5-aza-CdR and cyclic AMP (cAMP) reverse the CD44 gene silencing. Contrary to 5-aza-CdR, cAMP does not induce DNA demethylation or histone modification at the CD44 promoter, whereas an H3pS10/AcK14 dual modification is observed on a global level. cAMP also induces the expression of c-Jun transcription factor and its recruitment at the CD44 promoter. Chromatin immunoprecipitation assays further show the association of brahma (Brm), a subunit of SWI/SNF chromatin-remodelling complex involved in the crosstalk between transcription and RNA polymerase II (RNA Pol II) processing, as well as the binding of phosphorylated RNA Pol II to the proximal promoter region of CD44. Finally, our study reveals that cAMP re-establishes the CD44-mediated cell death signalling. We propose that one of the actions of cAMP in restoring normal cell phenotype of leukaemia cells may consist in a broad trans-reactivation of silenced genes, despite marked hypermethylation of their promoters, as illustrated here with CD44 re-expression.

Telomeres and telomerase: Pharmacological targets for new anticancer strategies?

Telomeres are located at the ends of eukaryotic chromosomes. Human telomerase, a cellular reverse transcriptase, is a ribonucleoprotein enzyme that catalyzes the synthesis and extension of telomeric DNA. It is composed of at least, a template RNA component (hTR; human Telomerase RNA) and a catalytic subunit, the telomerase reverse transcriptase (hTERT). The absence of telomerase is associated with telomere shortening and aging of somatic cells, while high telomerase activity is observed in over 85% of human cancer cells, strongly indicating its key role during tumorigenesis. Several details regarding telomere structure and telomerase regulation have already been elucidated, providing new targets for therapeutic exploitation. Further support for anti-telomerase approaches comes from recent studies indicating that telomerase is endowed of additional functions in the control of growth and survival of tumor cells that do not depend only on the ability of this enzyme to maintain telomere length. This observation suggests that inhibiting telomerase or its synthesis may have additional anti-proliferative and apoptosis inducing effect, independently of the reduction of telomere length during cell divisions. This article reviews the basic information about the biology of telomeres and telomerase and attempts to present various approaches that are currently under investigation to inhibit its expression and its activity. We summarize herein distinct anti-telomerase approaches like antisense strategies, reverse transcriptase inhibitors, and G-quadruplex interacting agents, and also review molecules targeting hTERT expression, such as retinoids and evaluate them for their therapeutic potential. "They conceive a certain theory, and everything has to fit into that theory. If one little fact will not fit it, they throw it aside. But it is always the facts that will not fit in that are significant". "Death on the Nile". Agatha Christie.

Telomerase targeting by retinoids in cells from patients with myeloid leukemias of various subtypes, not only APL.

Numerous strategies have been proposed to specifically inhibit telomerase (human telomerase reverse transcriptase (hTERT)) but to date only a few are clinically relevant in anticancer therapy. Recently, we have shown that long-term treatment with all-trans retinoic acid (ATRA), a compound clinically approved for differentiation therapy of acute promyelocytic leukemia (APL), represses hTERT in differentiation-resistant APL cell lines leading to telomere shortening and death. This signaling requires the co-activation of the retinoic acid receptor alpha (RARalpha) and the retinoic X receptor (RXR). In contrast to differentiation-therapy, which is only successful in this subtype of leukemia, the telomerase-targeted pathway could also be of use in non-APL. Here, we demonstrate that repression of hTERT occurs in fresh blasts cells from patients with myeloid leukemias of various subtypes exposed ex vivo to ATRA or synthetic retinoids. These results support the idea that, by hTERT targeting, retinoids can induce telomere shortening and cell death and their integration in therapy protocols for myeloid leukemias refractory to maturation should be considered.

Retinoid/arsenic combination therapy of promyelocytic leukemia: induction of telomerase-dependent cell death.

Acute promyelocytic leukemia (APL) is efficiently treated with a cell differentiation inducer, all-trans retinoic acid (ATRA). However, a significant percentage of patients still develop resistance to this treatment. Recently, arsenic trioxide (As2O3), alone or in combination with ATRA, has been identified as an alternative therapy in patients with both ATRA-sensitive and ATRA-resistant APL. Previous investigations restricted the mechanism of this synergism to the modulation and/or degradation of PML-RARalpha oncoprotein through distinct pathways. In this study, using several ATRA maturation-resistant APL cell lines, we demonstrate in vitro that the success of ATRA/As2O3 treatment in APL pathology can be explained, at least in part, by a synergistic effect of these two drugs in triggering downregulation of telomerase efficient enough to cause telomere shortening and subsequent cell death. Such long-term low-dose combinatorial therapy strategies, developed also to avoid acute side effects, reinforce the notion that the antitelomerase strategy, based on a combination of active agents, should now be considered and evaluated not only in APL but also in other malignancies.

Receptor heteromerization in adenosine A2A receptor signaling: relevance for striatal function and Parkinson's disease.

Recently evidence has been presented that adenosine A2A and dopamine D2 receptors form functional heteromeric receptor complexes as demonstrated in human neuroblastoma cells and mouse fibroblast Ltk- cells. These A2A/D2 heteromeric receptor complexes undergo coaggregation, cointernalization, and codesensitization on D2 or A2A receptor agonist treatments and especially after combined agonist treatment. It is hypothesized that the A2A/D2 receptor heteromer represents the molecular basis for the antagonistic A2A/D2 receptor interactions demonstrated at the biochemical and behavioral levels. Functional heteromeric complexes between A2A and metabotropic glutamate 5 receptors (mGluR5) have also recently been demonstrated in HEK-293 cells and rat striatal membrane preparations. The A2A/mGluR5 receptor heteromer may account for the synergism found after combined agonist treatments demonstrated in different in vitro and in vivo models. D2, A2A, and mGluR5 receptors are found together in the dendritic spines of the striatopallidal GABA neurons. Therefore, possible D2/A2A/mGluR5 multimeric receptor complexes and the receptor interactions within them may have a major role in controlling the dorsal and ventral striatopallidal GABA neurons involved in Parkinson's disease and in schizophrenia and drug addiction, respectively.

Interactions among adenosine deaminase, adenosine A(1) receptors and dopamine D(1) receptors in stably cotransfected fibroblast cells and neurons.

The role of adenosine deaminase in the interactions between adenosine A(1) and dopamine D(1) receptors was studied in a mouse fibroblast cell line stably cotransfected with human D(1) receptor and A(1) receptor cDNAs (A(1)D(1) cells). Confocal laser microscopy analysis showed a high degree of adenosine deaminase immunoreactivity on the membrane of the A(1)D(1) cells but not of the D(1) cells (only cotransfected with human D(1) receptor cDNAs). In double immunolabelling experiments in A(1)D(1) cells and cortical neurons a marked overlap in the distribution of the A(1) receptor and adenosine deaminase immunoreactivities and of the D(1) receptor and adenosine deaminase immunoreactivities was found. Quantitative analysis of A(1)D(1) cells showed that adenosine deaminase immunoreactivity to a large extent colocalizes with A(1) and D(1) receptor immunoreactivity, respectively. The A(1) receptor agonist caused in A(1)D(1) cells and in cortical neurons coaggregation of A(1) receptors and adenosine deaminase, and of D(1) receptors and adenosine deaminase. The A(1) receptor agonist-induced aggregation was blocked by R-deoxycoformycin, an irreversible adenosine deaminase inhibitor. The competitive binding experiments with the D(1) receptor antagonist [(3)H]SCH-23390 showed that the D(1) receptors had a better fit for two binding sites for dopamine, and treatment with the A(1) receptor agonist produced a disappearance of the high-affinity site for dopamine at the D(1) receptor. R-Deoxycoformycin treatment, which has previously been shown to block the interaction between adenosine deaminase and A(1) receptors, and which is crucial for the high-affinity state of the A(1) receptor, also blocked the A(1) receptor agonist-induced loss of high-affinity D(1) receptor binding. The conclusion of the present studies is that the high-affinity state of the A(1) receptor is essential for the A(1) receptor-mediated antagonistic modulation of D(1) receptors and for the A(1) receptor-induced coaggregates of A(1) and adenosine deaminase, and of D(1) and adenosine deaminase. Thus, the confocal experiments indicate that both A(1) and D(1) receptors form agonist-regulated clusters with adenosine deaminase, where the presence of a structurally intact adenosine deaminase bound to A(1) receptors is important for the A(1)-D(1) receptor-receptor interaction at the level of the D(1) receptor recognition.

All-trans retinoic acid down-regulates prion protein expression independently of granulocyte maturation.

The cellular prion protein (PrPc) is a sialoglycoprotein involved in the pathogenesis of prion diseases. It has been identified at the plasma membrane of several cell types. All-trans retinoic acid (ATRA) is known to induce differentiation of human leukemia cell lines in vitro. PrPc messenger ribonucleic acid (mRNA) and protein are down-regulated upon ATRA-induced differentiation of HL60 cells. In this report, we have investigated the regulation of PrPc mRNA and protein expression during ATRA-treatment of maturation-sensitive (NB4) and -resistant (NB4-R1 and NB4-R2) cell lines. In ATRA-induced maturation of NB4 cells, down-regulation of PrPc mRNA and protein were observed. We also show that down-regulation of PrPc mRNA is dependent on protein synthesis. Moreover, the same down-regulation of prion protein by ATRA was observed at the surface of maturation-resistant, ATRA-responsive NB4-R1 cells. In contrast, the maturation-resistant and ATRA-unresponsive NB4-R2 subline showed no variation in membrane prion protein expression. These results demonstrate a dissociation between the regulation of prion protein expression by ATRA and the process of granulocyte maturation. We propose that retinoids should be investigated further as a preventive strategy to slow down prion disease progression.

Autonomous rexinoid death signaling is suppressed by converging signaling pathways in immature leukemia cells.

On their own, retinoid X receptor (RXR)-selective ligands (rexinoids) are silent in retinoic acid receptor (RAR)-RXR heterodimers, and no selective rexinoid program has been described as yet in cellular systems. We report here on the rexinoid signaling capacity that triggers apoptosis of immature promyelocytic NB4 cells as a default pathway in the absence of survival factors. Rexinoid-induced apoptosis displays all features of bona fide programmed cell death and is inhibited by RXR, but not RAR antagonists. Several types of survival signals block rexinoid-induced apoptosis. RARalpha agonists switch the cellular response toward differentiation and induce the expression of antiapoptosis factors. Activation of the protein kinase A pathway in the presence of rexinoid agonists induces maturation and blocks immature cell apoptosis. Addition of nonretinoid serum factors also blocks cell death but does not induce cell differentiation. Rexinoid-induced apoptosis is linked to neither the presence nor stability of the promyelocytic leukemia-RARalpha fusion protein and operates also in non-acute promyelocytic leukemia cells. Together our results support a model according to which rexinoids activate in certain leukemia cells a default death pathway onto which several other signaling paradigms converge. This pathway is entirely distinct from that triggered by RAR agonists, which control cell maturation and postmaturation apoptosis.

Staurosporine induces apoptosis through both caspase-dependent and caspase-independent mechanisms.

Sensitivity of tumor cells to anticancer therapy depends on the ability of the drug to induce apoptosis. However, multiple signaling pathways control this induction and thus determine this sensitivity. We report here that staurosporine, a well known inducer of apoptosis in a wide range of cell lines, displays distinct ability to trigger apoptosis in two different L1210 sublines (termed L1210/S and L1210/0). Staurosporine treatment resulted in an early cell death (within 3 h) in L1210/S cells, while in L1210/0 cells, death occurred only after 12 h. In both instances, death occurred by apoptosis. A broad spectrum caspase inhibitor, Z-VAD-fmk, blocked early apoptosis in L1210/S cells but did not confer any protection on late apoptosis in L1210/0 cells. Protection by Z-VAD-fmk observed in L1210/S cells was not lasting and unmasked a secondary process of cell death that also exhibited characteristics of apoptosis. Thus, staurosporine induces apoptotic cell death through at least two redundant parallel pathways. These two pathways normally coexist in L1210/S cells. However, the early cell death mechanism depending on caspase activation disguises the late caspase-independent apoptotic process. Staurosporine-induced apoptosis in L1210/0 cells develops only by the caspase-independent mechanism due to a general defect in caspase activation.

Evidence for adenosine/dopamine receptor interactions: indications for heteromerization.

Evidence has been obtained for adenosine/dopamine interactions in the central nervous system. There exists an anatomical basis for the existence of functional interactions between adenosine A(1)R and dopamine D(1)R and between adenosine A(2A) and dopamine D(2) receptors in the same neurons. Selective A(1)R agonists affect negatively the high affinity binding of D(1) receptors. Activation of A(2A) receptors leads to a decrease in receptor affinity for dopamine agonists acting on D(2) receptors, specially of the high-affinity state. These interactions have been reproduced in cell lines and found to be of functional significance. Adenosine/dopamine interactions at the behavioral level probably reflect those found at the level of dopamine receptor binding and transduction. All these findings suggest receptor subtype-specific interactions between adenosine and dopamine receptors that may be achieved by molecular interactions (e.g., receptor heterodimerization). At the molecular level adenosine receptors can serve as a model for homomeric and heteromeric protein-protein interactions. A1R forms homodimers in membranes and also form high-order molecular structures containing also heterotrimeric G-proteins and adenosine deaminase. The occurrence of clustering also clearly suggests that G-protein- coupled receptors form high-order molecular structures, in which multimers of the receptors and probably other interacting proteins form functional complexes. In view of the occurrence of homodimers of adenosine and of dopamine receptors it is speculated that heterodimers between these receptors belonging to two different families of G-protein-coupled receptors can be formed. Evidence that A1/D1 can form heterodimers in cotransfected cells and in primary cultures of neurons has in fact been obtained. In the central nervous system direct and indirect receptor-receptor interactions via adaptor proteins participate in neurotransmission and neuromodulation and, for example, in the establishment of high neural functions such as learning and memory.

Dopamine D1 and adenosine A1 receptors form functionally interacting heteromeric complexes.

The possible molecular basis for the previously described antagonistic interactions between adenosine A(1) receptors (A(1)R) and dopamine D(1) receptors (D(1)R) in the brain have been studied in mouse fibroblast Ltk(-) cells cotransfected with human A(1)R and D(1)R cDNAs or with human A(1)R and dopamine D(2) receptor (long-form) (D(2)R) cDNAs and in cortical neurons in culture. A(1)R and D(1)R, but not A(1)R and D(2)R, were found to coimmunoprecipitate in cotransfected fibroblasts. This selective A(1)R/D(1)R heteromerization disappeared after pretreatment with the D(1)R agonist, but not after combined pretreatment with D(1)R and A(1)R agonists. A high degree of A(1)R and D(1)R colocalization, demonstrated in double immunofluorescence experiments with confocal laser microscopy, was found in both cotransfected fibroblast cells and cortical neurons in culture. On the other hand, a low degree of A(1)R and D(2)R colocalization was observed in cotransfected fibroblasts. Pretreatment with the A(1)R agonist caused coclustering (coaggregation) of A(1)R and D(1)R, which was blocked by combined pretreatment with the D(1)R and A(1)R agonists in both fibroblast cells and in cortical neurons in culture. Combined pretreatment with D(1)R and A(1)R agonists, but not with either one alone, substantially reduced the D(1)R agonist-induced accumulation of cAMP. The A(1)R/D(1)R heteromerization may be one molecular basis for the demonstrated antagonistic modulation of A(1)R of D(1)R receptor signaling in the brain. The persistence of A(1)R/D(1)R heteromerization seems to be essential for the blockade of A(1)R agonist-induced A(1)R/D(1)R coclustering and for the desensitization of the D(1)R agonist-induced cAMP accumulation seen on combined pretreatment with D(1)R and A(1)R agonists, which indicates a potential role of A(1)R/D(1)R heteromers also in desensitization mechanisms and receptor trafficking.

Ectopic expression of Bcl-2 switches over nuclear signalling for cAMP-induced apoptosis to granulocytic differentiation.

The IPC-81 myeloid leukaemia cells undergo apoptosis rapidly after cAMP stimulation (6 h) and cell death is prevented by early over-expression of the cAMP-inducible transcription repressor ICER, that blocks cAMP-dependent nuclear signalling. Therefore, the expression of specific genes controlled by CRE-containing promoters is likely to determine cell fate. We now show that cAMP-induced cell death also is abrogated by the over-expression of the anti-apoptotic gene, Bcl-2. Contrary to ICER, Bcl-2 does not affect cAMP-signalling and allows the analysis of cAMP responses in death rescued cells. The Bcl-2 transfected cells treated with 8-CPT-cAMP were growth-arrested and thereafter cells embarked in granulocytic differentiation, with no additional stimulation. Neutrophilic polynuclear granulocytes benefited from a long life span in G0-G1 and remained functional (phagocytosis). This work demonstrates that, using anti-apoptosis regulators, 'death signals' could be exploited to trigger distinct biological responses. Indeed, cAMP signal can trigger several simultaneously developing biological programs, in the same cell, i.e., growth regulation, apoptosis and differentiation. This cell system should prove useful to determine how a tumour cell can be re-programmed for either apoptosis or functional maturation by physiological signals.

A stereological study on the neuroprotective actions of acute modafinil treatment on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced nigral lesions of the male black mouse.

The effect of an acute administration of the vigilance-promoting drug modafinil ((+/-)(diphenyl-methyl)-sulfinyl-2 acetamide; Modiodal) on the nigrostriatal dopamine system was studied after damage induced by MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) by means of immunohistochemistry for tyrosine hydroxylase (TH) and a stereological method. MPTP (40 mg/kg) reduced from 24,380 +/- 902 to 13,501 +/- 522 and from 37,868 +/- 3300 to 20,568 +/- 1270, respectively, the number of TH immunoreactive (IR) and non-TH IR nigral neurons. Co-administration of Modafinil restored to normal the number of these neuronal populations. MPTP treatment induced also a reduction in the volume of TH IR neurons, which was counteracted by Modafinil administration. The data provide morphological evidence, based on unbiased stereological analysis, for a potential neuroprotective role of Modafinil, not only in dopaminergic neurons, but also with a similar magnitude in the non-DA nerve cell population of the substantia nigra after MPTP lesion. These results suggest that Modafinil has a neuroprotective role in the substantia nigra via a still undefined mechanism in which a crucial role of DA uptake blockade should be excluded. Modafinil may therefore have a therapeutic potential in neurodegenerative processes such as those occurring in Parkinson's disease.

MLL-AF1q fusion resulting from t(1;11) in acute leukemia.

The MLL gene, located on chromosome band 11q23 is fused to different partner genes as a result of various chromosomal translocations in hematopoietic malignancies. A t(1;11) (q21;q23) resulting in a MLL-AF1q fusion gene has previously been reported. Cytogenetic studies on six cases are reported, including one three-way translocation. FISH analysis using a YAC encompassing the MLL gene and a YAC encompassing the AF1q locus showed splitting in three cases and two patients, respectively. PCR analysis of two cases confirmed that AF1q is specifically associated with t(1;11)(q21;q23). The MLL-AF1q fusion mRNA was similar to that previously described in one case and involved MLL exon 7 in the other. This study confirms the specific involvement of AF1q in t(1;11) (q21;q23)-positive acute leukemia with monocytic involvement.

A new case of translocation t(6;11)(q21;q23) in a therapy-related acute myeloid leukemia resulting in an MLL-AF6q21 fusion.

A new case of translocation t(6;11)(q21;q23) in a patient with therapy-related acute myeloblastic leukemia is reported. The translocation results in fusion of the MLL and AF6q21 genes. The breakpoint with AF6q21 is located within the sequences encoding the AF6q21 fork head motif. The similar location of the localization of the chromosome 6 breakpoints in the present case and in the first case reported suggests their nonrandom localization. In addition, treatment for Hodgkin's disease prior to leukemia in both t(6;11)(q21;q23) cases suggests an association of this translocation with therapy-related leukemias, as reported for the recently described.

Modafinil prevents glutamate cytotoxicity in cultured cortical neurons.

The ability of modafinil (Modiodal) to protect cortical neurons from glutamate-induced degeneration was evaluated by measuring electrically evoked [3H]GABA release and [3H]GABA uptake in primary cerebral cortical cultures. In normal cells, electrical stimulation (10 Hz, 2 min) increased [3H]GABA release (FR-NER St1 = 0.77+/-0.14; St2/St1 ratio = 0.94+/-0.02). The exposure of sister cells to glutamate, reduced electrically evoked [3H]GABA release (FR-NER St1 = 0.40+/-0.05; St2/St1 ratio = 0.60+/-0.08). Modafinil (0.3-1 microM) prevented the glutamate-induced reduction of the St2/St1 ratio (0.85+/-0.11; 0.88+/-0.05, respectively). A similar protective effect was observed for [3H]GABA uptake. These findings suggest that modafinil may be neuroprotective in that it attenuates glutamate-induced excitotoxicity in cortical neurons.

AF6q21, a novel partner of the MLL gene in t(6;11)(q21;q23), defines a forkhead transcriptional factor subfamily.

Fusion genes implicating the MLL gene have been recently demonstrated in various 11q23 chromosomal abnormalities in human hematopoietic malignancies. We analyzed a t(6;11)(q21;q23) translocation detected in a secondary acute myeloblastic leukemia. This translocation results in fusion of the MLL gene on 11q23 to a previously unknown gene on chromosome 6 that differs from the previously reported MLL partner gene AF6q. The novel gene, named AF6q21, encodes a forkhead (FH) protein with strong similarities to the two FH family members whose genes are already known to be involved in chromosomal translocations of human malignancies, AFX and FKHR. Strikingly, in these translocations the breakpoints are located at the same position within the FH domains. Therefore, AF6q21, AFX, and FKHR could define a new FH subfamily particularly involved in human malignancies.

A novel class of zinc finger/leucine zipper genes identified from the molecular cloning of the t(10;11) translocation in acute leukemia.

A novel class of conserved transcription factors has been identified from the molecular cloning of AF10, the gene involved in the t(10;11)(p12;q23) translocation of acute myeloid leukemias. AF10 encodes a 109-kD protein of 1,027 amino acids and contains an N-terminal zinc finger region and a C-terminal leucine zipper. These structures have been found to be conserved in sequence and position in three other proteins, AF17, BR140, and a previously unrecognized Caenorhabditis elegans gene, provisionally named CEZF. The overall structure, level of sequence conservation, and expression pattern suggest that these genes encode a new class of transcription factors, some of which are targets for chromosomal translocation in acute leukemia.

Effects of 50 Hz magnetic fields on C-myc transcript levels in nonsynchronized and synchronized human cells.

The effects of 50 Hz electromagnetic fields (EMFs) on the expression of the c-myc oncogene, known to be involved in normal cell proliferation and possibly also in tumor processes, were investigated in nonsynchronized human lymphoid cells immortalized by Epstein-Barr virus. Viral injury to such cells makes them a good model for exploring the possible cancer-promoting effects of 50 Hz magnetic fields. Parallel experiments were conducted on human HL60 leukemic cells. Cells were exposed to sinusoidal 50 Hz EMFs at 10 microT or 1 mT for 20 min, 1 h, 24 h, or 72 h. Exposure was performed either immediately after refeeding or 1.5 h after refeeding. C-myc transcript values were assessed by Northern blot analysis and normalized to those of the noninducible gene GaPDH. No statistically significant difference between the c-myc transcript levels of control and exposed cells was found in lymphoid or leukemic cells under our experimental conditions, either after short exposures of 20 min and 1 h or after longer exposures of 24 and 72 h. Other experiments were carried out with pseudosynchronized cells in an attempt to establish whether cells were especially sensitive to 50 Hz magnetic field exposure in any particular phase of the cell cycle. Accordingly, cells were pseudosynchronized in G0/G1 by serum deprivation and exposed for 20 min to a 50 Hz magnetic field, at 10 microT for lymphoid cells and 1 mT for HL60 cells. No significant difference was observed between the c-myc transcript levels of control and exposed cells for either of the synchronized cell types. These results for synchronized cells correlated with those for nonsynchronized cells.