Annarosa Arcangeli

herg1 Gene and HERG1 Protein Are Overexpressed in Colorectal Cancers and Regulate Cell Invasion of Tumor Cells

The acquisition of the capacity to invade surrounding tissues confers a more malignant phenotype to tumor cells and is necessary for the establishment of metastases. The understanding of the molecular mechanisms underlying cell invasion in human solid tumors such as colorectal cancers could provide not only more sensitive prognostic analyses but also novel molecular targets for cancer therapy. We report in this article that K+ ion channels belonging to the HERG family are important determinants for the acquisition of an invasive phenotype in colorectal cancers. The herg1 gene and HERG1 protein are expressed in many colon cancer cell lines, and the activity of HERG channels modulates colon cancer cell invasiveness. Moreover, the amount of HERG1 protein expressed on the plasma membrane is directly related to the invasive phenotype of colon cancer cells. Finally, both the herg1 gene and HERG1 protein were expressed in a high percentage of primary human colorectal cancers, with the highest incidence occurring in metastatic cancers, whereas no expression could be detected either in normal colonic mucosa or in adenomas.

Cell Cycle-dependent Expression of HERG1 and HERG1B Isoforms in Tumor Cells

The role of K+ channel activity during cell cycle progression has become a research topic of considerable interest. Blocking of K+ channels inhibits the proliferation of many cell types, although the mechanism of this inhibition is unclear. There is speculation that K+channels differentially regulate the electrical potential of the plasma membrane (V m ) during proliferation. We have demonstrated that in tumor cells the value of V m is clamped to rather depolarized values by K+ channels belonging to the HERG family. We report here that tumor cell lines preferentially express the herg1 gene and a truncated,N-deleted form that corresponds to herg1b. This alternative transcript is also expressed in human primary acute myeloid leukemias. Both HERG1 and HERG1B proteins are expressed on the plasma membrane of tumor cells and can form heterotetramers. The expression of HERG protein isoforms is strongly cell cycle-dependent, accounting for variations in HERG currents along the mitotic cycle. Moreover, the blocking of HERG channels dramatically impairs cell growth of HERG-bearing tumor cells. These results suggest that modulated expression of different K+ channels is the molecular basis of a novel mechanism regulating neoplastic cell proliferation.

HERG potassium channels are constitutively expressed in primary human acute myeloid leukemias and regulate cell proliferation of normal and leukemic hemopoietic progenitors.

An important target in the understanding of the pathogenesis of acute myeloid leukemias (AML) relies on deciphering the molecular features of normal and leukemic hemopoietic progenitors. In particular, the analysis of the mechanisms involved in the regulation of cell proliferation is decisive for the establishment of new targeted therapies. To gain further insight into this topic we report herein a novel approach by analyzing the role of HERG K+ channels in the regulation of hemopoietic cell proliferation. These channels, encoded by the human ether-a-gò-gò-related gene (herg), belong to a family of K+ channels, whose role in oncogenesis has been recently demonstrated. We report here that herg is switched off in normal peripheral blood mononuclear cells (PBMNC) as well as in circulating CD34+ cells, however, it is rapidly turned on in the latter upon induction of the mitotic cycle. Moreover, hergappears to be constitutively activated in leukemic cell lines as well as in the majority of circulating blasts from primary AML. Evidence is also provided that HERG channel activity regulates cell proliferation in stimulated CD34+ as well as in blast cells from AML patients. These results open new perspectives on the pathogenetic role of HERG K+ channels in leukemias.

A novel role for HERG K+ channels: spike-frequency adaptation.

1 The regular firing of a Hodgkin‐Huxley neurone endowed with fast Na+ and delayed K+ channels can be converted into adapting firing by appending HERG (human eag‐related gene) channels. 2 The computer model predictions were verified by studying the firing properties of F‐11 DRG neurone x neuroblastoma hybrid cells induced to differentiate by long‐term exposure to retinoic acid. These cells, which express HERG currents (IHERG), show clear spike‐frequency adaptation of their firing when current clamped with long depolarizations. 3 In agreement with the prediction, the selective blocking of IHERG by class III antiarrhythmic drugs always led to the disappearance of the spike‐frequency adaptation, and the conversion of adapting firing to regular firing. 4 It is proposed that, in addition to their role in the repolarization of the heart action potential, HERG channels may sustain a process of spike‐frequency adaptation, and hence contribute to the control of burst duration in a way that is similar to that of the K+ currents, IAHP, IC and IM. In addition to the known cardiac arrhythmia syndrome (LQT2), genetic mutations or an altered HERG expression could lead to continuous hyperexcitable states sustained by the inability of nerve or endocrine cells to accommodate to repetitive stimuli. This might help in clarifying the pathogenesis of still undefined idiopathic familial epilepsies.

Blockade of HERG potassium currents by fluvoxamine: incomplete attenuation by S6 mutations at F656 or Y652

Pharmacological blockade of the Human ether‐a‐go‐go related gene (HERG) potassium channel is commonly linked with acquired long QT syndrome and associated proarrhythmia. The objectives of this study were (i) to identify and characterise any inhibitory action on HERG of the selective‐serotonin re‐uptake inhibitor fluvoxamine, (ii) to then determine whether fluvoxamine shared the consensus molecular determinants of HERG blockade of those drugs so far tested. Heterologous HERG potassium current (IHERG) was measured at 37°C, using the whole‐cell patch‐clamp technique, from a mammalian cell line (Human embryonic kidney 293) expressing HERG channels. IHERG tails, following repolarisation from +20 to −40 mV, were blocked by fluvoxamine with an IC50 of 3.8 μM. Blockade of wild‐type HERG was of extremely rapid onset (within 10 ms) and showed voltage dependence, with fluvoxamine also inducing a leftward shift in voltage‐dependent activation of IHERG. Characteristics of block were consistent with a component of closed channel (or extremely rapidly developing open channel) blockade and dependence on open and inactivated channel states. The attenuated‐inactivation mutation S631A partially reduced the blocking effect of fluvoxamine. The S6 mutations, Y652A and F656A, and the pore helix mutant S631A only partially attenuated blockade by fluvoxamine at concentrations causing profound blockade of wild‐type HERG. All HERG‐blocking pharmaceuticals studied to date have been shown to block F656 mutant channels with over 100‐fold reduced potency compared to their blockade of the wild‐type channel. Fluvoxamine is therefore quite distinct in this regard from previously studied agents.

HERG potassium channels are more frequently expressed in human endometrial cancer as compared to non-cancerous endometrium

HERG K+channels, besides contributing to regulate cardiac and neuronal excitability, are preferentially expressed in tumour cell lines of different histogenesis, where their role in the development and maintenance of the neoplastic phenotype is under study. We show here that both herg gene and HERG protein are expressed with high frequency in primary human endometrial cancers, as compared to normal and hyperplastic endometrium. RT-PCR and immunohistochemistry, using specific anti-HERG antibodies developed in our laboratory, were applied to tissue specimens obtained from 18 endometrial cancers and 11 non-cancerous endometrial tissues. herg RNA and HERG protein are expressed in 67% and 82%, respectively, of cancerous, while in only 18% of non-cancerous tissues. In particular, no expression was found in endometrial hyperplasia. Moreover, electrophysiological experiments confirmed the presence of functioning HERG channels on the plasma membrane of tumour cells. On the whole, these data are the first demonstration of the presence of HERG channels in primary human neoplasias, and could candidate HERG as a potential tool capable of marking cancerous versus hyperplastic endometrial growth.

Glucose- and arginine-induced insulin secretion by human pancreatic {beta}-cells: the role of HERG K+ channels in firing and release

The human ether‐a‐go‐go‐related genes (herg) are expressed in tissues other than heart and brain where the HERG K+ channels are known to regulate the repolarization of the heart action potential and the neuronal spike‐frequency accommodation. We provide evidence that herg1 transcripts are present in human pancreatic islets that were used to study both insulin secretion and electrical activity with radioimmunoassay and single cell perforated patch‐clamp techniques, respectively. Glucose‐ and arginine‐induced islets insulin secretion data suggested a net increase of release under perfusion with antiarrhythmic drugs known to selectively block HERG channels. Indeed we could routinely isolate a K+ current that was recognized as biophysically and pharmacologically similar to the HERG current. An analysis of the glucose‐ and arginine‐induced electrical activity (several applications during 30 min) in terms of firing frequency and putative insulin release was done in control and in the presence of selective blockers of HERG channels: the firing frequency and the release increased by 32% and 77%, respectively. It is concluded that HERG channels have a crucial role in regulating insulin secretion and firing of human β‐cells. This raises the possibility that some genetically characterized hyperinsulinemic diseases of unknown origin might involve mutations in the HERG channels.—Rosati, B., Marchetti, P., Crociani, O., Lecchi, M., Lupi, R., Arcangeli, A., Olivotto, M., Wanke, E. Glucose‐ and arginine‐in‐duced insulin secretion by human pancreatic β‐cells: the role of HERG K+ channels in firing and release. The FASEB J. 14, 2601–2610 (2000)

HERG‐ and IRK‐like Inward Rectifier Currents are Sequentially Expressed During Neuronal Development of Neural Crest Cells and their Derivatives

Quail neural crest cells were cultured in a differentiative medium to study the inward K+ channel profile in neuronal precursors at various stages of maturation. Between 12 and 24 h of culture, neural crest‐derived neurons displayed, in addition to the previously described outward depolarization‐activated K+ currents, an inward current enhanced in high K+ medium. A biophysical and pharmacological analysis led us to conclude that this inward K+ current is identical to that previously demonstrated in mouse and human neuroblastoma cell lines (IIR). This current (quail IIR or qllR), which is active at membrane potentials positive to ‐35 mV, was blocked by Cs+ and by class Ill antiarrhythmic drugs, thus resembling the K+ current encoded by the human ether‐a‐go‐go‐related gene (HERG). At later stages of incubation (>48 h), neural crest‐derived neurons underwent morphological and biochemical differentiation and expressed fast Na+ currents. At this stage the cells lost qllR, displaying instead a classical inward rectifier K+ (IRK) current (quail IIRK= qIIRK). This substitution was reflected in the resting potential (VREST), which became hyperpolarized by >20 mV compared with the 24 h cells. Neurons were also harvested from peripheral ganglia and other derivatives originating from the migration of neural crest cells, viz. ciliary ganglia, dorsal root ganglia, adrenal medulla and sympathetic chain ganglia. After brief culture following harvesting from young embryos, ganglionic neurons always expressed qilR. On the other hand, when ganglia were explanted from older embryos (7–12 days), briefly cultured neurons displayed the IRK‐like current. Again, in all the above derivatives the qllR substitution by qllRK was accompanied by a 20 mV hyperpolarization of VREST. Together, these data indicate that the VREST of normal neuronal precursors is sequentially regulated by HERG‐ and IRK‐like currents, suggesting that HERG‐like channels mark an immature and transient stage of neuronal differentiation, probably the same stage frozen in neuroblastomas by neoplastic transformation.

hERG1 channels are overexpressed in glioblastoma multiforme and modulate VEGF secretion in glioblastoma cell lines

Recent studies have led to considerable advancement in our understanding of the molecular mechanisms that underlie the relentless cell growth and invasiveness of human gliomas. Partial understanding of these mechanisms has (1) improved the classification for gliomas, by identifying prognostic subgroups, and (2) pointed to novel potential therapeutic targets. Some classes of ion channels have turned out to be involved in the pathogenesis and malignancy of gliomas. We studied the expression and properties of K+ channels in primary cultures obtained from surgical specimens: human ether a gò-gò related (hERG)1 voltage-dependent K+ channels, which have been found to be overexpressed in various human cancers, and human ether a gò-gò-like 2 channels, that share many of hERG1s biophysical features. The expression pattern of these two channels was compared to that of the classical inward rectifying K+ channels, IRK, that are widely expressed in astrocytic cells and classically considered a marker of astrocytic differentiation. In our study, hERG1 was found to be specifically overexpressed in high-grade astrocytomas, that is, glioblastoma multiforme (GBM). In addition, we present evidence that, in GBM cell lines, hERG1 channel activity actively contributes to malignancy by promoting vascular endothelial growth factor secretion, thus stimulating the neoangiogenesis typical of high-grade gliomas. Our data provide important confirmation for studies proposing the hERG1 channel as a molecular marker of tumour progression and a possible target for novel anticancer therapies.

A toxin to nervous, cardiac, and endocrine ERG K+ channels isolated from Centruroides noxius scorpion venom

Toxins isolated from a variety of venoms are tools for probing the physiological function and structure of ion channels. The ether‐a‐go‐go‐related genes (erg) codify for the K+ channels (ERG), which are crucial in neurons and are impaired in human long‐QT syndrome and Drosophila ‘seizure’ mutants. We have isolated a peptide from the scorpion Centruroides noxius Hoffmann that has no sequence homologies with other toxins, and demonstrate that it specifically inhibits (IC50 = 16±1 nM) only ERG channels of different species and distinct histogenesis. These results open up the possibility of investigating ERG channel structure‐function relationships and novel pharmacological tools with potential therapeutic efficacy.—Gurrola, G. B., Rosati, B., Rocchetti, M., Pimienta, G., Zaza, A., Arcangeli, A., Olivotto, M., Possani, L. D., Wanke, E. A toxin to nervous, cardiac, and endocrine ERG K+ channels isolated from Centruroides noxius scorpion venom. FASEB J. 13, 953–962 (1999)