Biochimie 82 (2000) 927−936 © 2000 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS. All rights reserved. S0300908400011652/REV
Peptide neurotoxins, small-cell lung carcinoma and neurological paraneoplastic syndromes Emanuele Shera*, Federica Giovanninib, John Boota, Betham Langb a
Lilly Research Centre, Erl Wood Manor, Sunninghill Road, Windlesham, Surrey, GU20 6PH, UK b Neurosciences Group, Institute of Molecular Medicine, J. Radcliffe Hospital, Oxford, UK (Received 29 March 2000; accepted 20 July 2000)
Abstract — Peptide neurotoxins isolated from the venom of snakes, spiders and snails have represented invaluable tools for the identification and characterisation of membrane ion channels and receptors in vertebrate cells, including human neurons. We here report on the use of these toxins for the characterisation of membrane ion channels and receptors expressed by one of the most aggressive human cancers, small-cell lung carcinoma. This tumour shares many properties with other neuro-endocrine cell types, including the ability of firing action potentials and release hormones in a calcium-dependent manner. Toxins such as α-bungarotoxin and x-conotoxins, among others, have been successfully used to characterise neuronal nicotinic receptors and voltage-dependent calcium channels, respectively, in human small-cell lung carcinoma cells. These receptors and ion channels are not only crucial for the growth of this specific tumour, but also represent autoantigens against which cancer patients build an autoimmune response. Although the aim of this autoimmune response is eventually the destruction of the cancer cells, the circulating antibodies cross-react with similar ion channels and receptors present in normal neurons or other cells, causing a number of different paraneoplastic diseases, the best characterised of which is the Lambert-Eaton myasthenic syndrome. Conotoxin-based radioimmunoassays have become an invaluable tool for the diagnosis and follow up of these paraneoplastic disorders and could represent a step forward in the early diagnosis of small-cell lung carcinoma itself. © 2000 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS small-cell lung carcinoma / bungarotoxin / conotoxin / myasthenic syndrome
1. Introduction Peptide neurotoxins have been amazing tools in biological research over the last three decades. Their selectivity and, in some cases, potency allowed for their use as ‘hooks’ to first identify and then characterise, purify and clone several ion channels of the vertebrate nervous system and they have been used by neuroscientists for studying the most intriguing neuronal cells. The availability of snake toxins, such as α-bungarotoxin, that bind to muscle nicotinic receptors, * Correspondence and reprints. E-mail address:
[email protected] (Emanuele Sher). Abbreviations: SCLC, small-cell lung carcinoma; nAchR, nicotinic acetylcholine receptor; VDCC, voltage-dependent calcium channel; VDKC, voltage-dependent potassium channel; DHP, dihydropyridine; TPMP, triphenyl-methyl-phosphonium; LEMS, Lambert-Eaton myasthenic syndrome; MG, myasthenia gravis; PCD, paraneoplastic cerebellar degeneration; α-Bgtx, α-bungarotoxin; j-Bgtx, j-bungarotoxin; α-Ctx MI, α-conotoxin form Conus magus, fraction I; α-Ctx ImI, α-conotoxin from Conus imperialis, fraction I; x-Ctx GVIA, x-conotoxin from Conus geographus, fraction VIA; x-Ctx MVIIC, x-conotoxin from Conus magus, fraction VIIC; x-Aga IVA, x-agatoxin from Agelenopsis aperta, fraction IVA. IgG, immunoglobulin G.
allowed the characterisation of this ‘prototype’ ion channel. Furthermore, the availability of this toxin was also instrumental in the dramatic development of studies on myasthenia gravis (MG), the best understood of the autoimmune disorders known to effect ion channels (for an historical prospective on these subjects, see [1, 2]). Recent evidence however, for which peptide neurotoxins are partially responsible, has slightly modified our views on the ‘peculiarity’ of nerve cells. Neurons seem to have added very specialised functions (and protein isoforms to accomplish them) to a common theme present in all excitable and non-excitable cells. For example, similar membrane ion channels and proteins of the secretory apparatus are shared by neurons, their ‘cousins’, the endocrine cells, as well by other, less related, epithelial or mesenchimal cells. This is the context into which we would like to consider the ‘peculiarity’ of small-cell carcinoma and related paraneoplastic disorders. SCLC is composed of epithelial cells with a ‘neuroendocrine’ phenotype. They are probably derived from the ‘neuroendocrine’ lung cells that are present either scattered or aggregated in the so-called neuroendocrine bodies in the bronchial mucosa, although this derivation is not yet formally proven. SCLC cells are characterised morphologically by the presence of cyto-
928 plasmic secretory granules that contain readily releasable peptides and amines [3]. They are also ‘excitable’, being able to generate spontaneous action potentials to which both Ca2 +, Na+ and K+ currents contribute [4–6]. Although excitation-secretion coupling has been studied thoroughly in both neurons, adrenal chromaffin cells and other endocrine cells, it is only recently that similar studies have been carried out on SCLC. SCLC are also often associated with paraneoplastic syndromes. These are disorders associated with tumours but not caused by the physical presence of the tumour. These include LambertEaton myasthenic syndrome (LEMS), subacute sensory neuronopathy and various forms of paraneoplastic encephalitis. A better understanding of excitation-secretion coupling in SCLC could be very helpful for at least two reasons: i) by modulating this process we could interfere with the biological activity of some of the released substances, crucial players in sustaining the autocrine stimulation of tumour growth, on one hand, and in causing peripheral, hormone-mediated, syndromes, on the other; and ii) autoimmune responses to proteins of the excitation-secretion apparatus present in SCLC can cause neurological and endocrine syndromes by attacking similar proteins present in nerve terminals and endocrine cells. Diagnostic and therapeutic improvements may be achieved. The aim of this review is to highlight the important role that peptide neurotoxins already had and can have in the future, in accomplishing some of the goals mentioned above. 2. Nicotinic neurotoxins and SCLC Although SCLC has long been recognised to be the tumour most often associated with tobacco smoking, research on the possible direct effects of nicotine on the biology of this cancer have been appearing only very recently. We will summarise below our current knowledge on nicotinic effects in SCLC cells, highlighting the role that peptide neurotoxins had in this research. 2.1. α-Bungarotoxin The first study carried out by Lennon and co-workers was unable to show the presence of AchR by immunoprecipitation with MG patients sera of 125I-α-BuTx-labelled proteins extracted from SCLC. However in the same study these authors were able to demonstrate the presence of muscarinic AchR [7]. The field remained relatively quiet until both Maneckjee and Minna [8] and Sher and colleagues [9] showed direct binding of 125I-α-Bgtx to human SCLC cell lines as well as to a number of other tumoural cell lines and fresh SCLC biopsies. At the same time, binding of other ligands such as 3H-nicotine [8] and 3 H-TPMP [10], supported the evidence that nicotinic receptors were indeed present in SCLC cells.
Sher et al. The question then moved to the molecular identity of these α-Bgtx-binding receptors. Were they muscle-type nicotinic receptors, or were they the so-called ‘neuronal α-Bgtx receptors of unknown function’ as were defined at that time? The inability of both MG sera and mAb35, a monoclonal antibody that recognises the muscle nicotinic receptor, to immunoprecipitate the 125I-αBgtx-labelled proteins was strong evidence against the AChR being of the muscle isotype. Furthermore, mRNA encoding for the α1, muscle specific, nicotinic subunit was not detected in these cells [9]. However, we found that the SCLC α-Bgtx receptor was immunoprecipitated by a polyclonal antiserum raised against the neuronal α-Bgtx receptor [9] present in IMR32 human neuroblastoma cells [11]. This fact first suggested the ‘neuronal’ nature of the SCLC receptors. Following, the discovery that neuronal α-Bgtx receptors were indeed nicotinic ion channels encoded by the α7 gene was published [12], and we were able to test whether α7 mRNA was present in SCLC cells and this was indeed the case [13]. A molecular identity for the SCLC α-Bgtx receptors was found. Another ‘neuronal’ marker was identified for SCLC cells. A critical role of these α-Bgtx-sensitive α7 nicotinic receptors in SCLC biology was shortly later discovered. We had previously demonstrated that nicotine and cytisine were able to stimulate both serotonin release and cell proliferation in human SCLC cell lines [14], both effects being antagonised by the non-selective nicotinic antagonist mecamylamine. Similarly, both serotonin release and cell proliferation were also antagonised by α-Bgtx, implicating the α7-containing receptors in these two crucial events [13]. These results were independently confirmed by both Quick et al. [15] and Schuller and Orloff [16]. α-Bgtx-sensitive α7 nicotinic receptors can increase intracellular calcium levels by two different mechanisms: they are highly calcium permeable and they can also depolarise the cells sufficiently to trigger the opening of voltage-sensitive sodium and calcium channels. They have also been implicated in the potentiation of glutamatergic synaptic transmission [17] as well as in the stimulation of catecholamine release from adrenal chromaffin cells [18] and may serve a very similar role in SCLC cells. However, in these cells, this activity leads also to tumour cell proliferation, possibly because of the combined effects of intracellular calcium increase and of the mitogenic properties of the released hormones. Could α7 antagonists be useful to prevent or treat SCLC? An important question remains open: are the α7 nicotinic receptors the only nicotinic receptors expressed in SCLC cells? The answer is probably no. We found that SCLC also express other ‘neuronal’ nicotinic subunits, namely the α3, α5, β2 and β4 subunits [9, 10] typically found, together with α7, in peripheral neurons. Lennon and co-workers did recently report that certain cloned
Peptide neurotoxins SCLC cells lines may express the muscle α1 subunit [19]. Very little is known about the physio-pathological role of these non-α7 receptors in SCLC. Hopefully, the use of new and highly selective peptide ‘neurotoxins’ will help in elucidating these issues. 2.2. j-Bungarotoxin Another peptide neurotoxin purified from the venom of Bungarus multicinctus is j-bungarotoxin which targets nicotinic receptors but with a different specificity to α-Bgtx. For some time, the contamination of ‘purified’ α-Bgtx with residual j-Bgtx (they have a very similar molecular mass) caused the generation of conflicting results on the specificity and functional effects of these two toxins. There is now general agreement that while α-Bgtx binds selectively to α1, α7, α8 and α9 among the known nicotinic subunits, j-Bgtx binds preferentially to α3 especially when co-assembled with β2 [20]. However at higher concentrations j-Bgtx will bind also to α1 and α7. When the identity and functional role of the α-Bgtx-sensitive receptors was not yet known, functional neuronal receptors were used to be classified as those at which j-Bgtx selectively bound. More specifically, when performing binding assays on neuronal membranes with 125I-j-Bgtx (one finds that) part of this binding can be displaced by α-Bgtx (i.e., the binding to α7 receptor) whilst the remainder is undisplaceable by α-Bgtx (α3-containing receptors). These two components (α3- and α7-receptors) of 125I-j-Bgtx binding can be expressed at different proportions in different neurons. We found that 125I-j-Bgtx binds to human SCLC cell membranes [21]. This binding, at least in GLC8 cells, is mostly (80%) antagonised by cold α-Bgtx, suggesting it is on the α7 receptors. However there is a small component of 125I-j-Bgtx that is not displaceable by α-Bgtx and this is likely to be α3 receptors. We cannot exclude at the present time that in other SCLC cell lines, or in vivo, different proportions of the two receptor subtypes could be present. j-Bgtx did antagonise nicotine-induced secretion and cell proliferation in GLC8 cells, but because, at least in these cells, the majority of j-Bgtx binding is on α7, this finding, more than suggesting a role for α3, corroborates a dominant role for α7 in mediating these effects. 2.3. α-Conotoxin MI α-Ctx MI (isolated from Conus magus) is one member of the growing family of small peptide toxins present in the venom of Conus marine snails that specifically target nicotinic receptors [22]. It has been shown to bind to α1 muscle-type receptors from many different species. More interesting, α-Ctx MI has been shown to bind with very
929 different affinity to the two non-equivalent acetylcholine binding sites formed at the α/γ or α/δ subunits interfaces, representing a powerful tool for studying the molecular pharmacology of these receptors. Also intriguing, is the finding that the selectivity of α-Ctx MI for the two acetylcholine binding sites is species-specific, being opposite in receptors derived from the electric ray Torpedo as compared to those from mammalian sources [23]. α-Ctx MI has been tested for its activity on neuronal nicotinic receptors. α-Ctx MI did not bind to rat neuronal receptors expressed in oocytes, confirming its selectivity for α1 muscle-type receptors [20]. However, α7 receptors were not evaluated in that report. Surprisingly, α-Ctx MI did block both serotonin release and cell proliferation in a number of different SCLC cell lines [13] More work has to be done to determine α-Ctx MI specificity for human neuronal nicotinic receptors, including α7, especially because of the species-specificity of some activities of this toxin. However, our finding strongly suggest that α-Ctx MI binds to human α7 receptors at least in SCLC cells. However, since α-Ctx MI has previously been shown to be selective for α1 receptors, and in light of the recent report that few SCLC cells could express α1, one could imagine that α-Ctx MI effects in SCLC could be mediated by these α1 receptors, possibly present at very low levels in the cells used. We consider this a very unlikely possibility but further experiments will help in solving this issue. 2.4. α-Conotoxin ImI α-Ctx ImI is another conopeptide isolated from the venom of Conus imperialis. It has been reported to have a specificity opposite to that of α-Ctx MI (and indeed also α-conotoxins GI, SI, EI etc.), being highly selective for α7 versus α1 receptors. This has been shown in both recombinant receptors [24] and by the ability of α-Ctx ImI to inhibit α7-mediated, α-Bgtx-sensitive currents in hippocampal neurons [25]. In our hands, α-Ctx ImI was found to be a potent antagonist of both nicotine-induced serotonin release and cell proliferation in three different human SCLC cell lines [26]. This finding not only confirms our previous demonstration of a dominant role of α7 in mediating nicotine effects on SCLC cells, but also suggests that α-Ctx ImI could become a useful tool also for studying human α7. We are not aware of any more recent studies evaluating α-Ctx ImI effects on human α7 in systems other than SCLC cells. 3. VDCC neurotoxins and SCLC As mentioned above, SCLC cells can fire action potentials with an important calcium component. Depolarisation (high KCl)-induced influx of radioactive calcium in human SCLC cells has been demonstrated first by Roberts
930 et al. [27] and then by others [28]. Sher et al. later reported Fura-2 measurements of intracellular calcium increases in response to high KCl [29]. These results support the hypothesis that SCLC express functionally active VDCCs. In the first studies, only dihydropyridine-sensitive (L-type VDCC) calcium fluxes were clearly demonstrated in SCLC cells. Indeed, at that time, only L-type channels were supposed to be present in the periphery, with VDCC heterogeneity remaining, for the moment, confined to neuronal cells. The discovery of peptide neurotoxins has revolutionised also our knowledge about the subtypes of VDCCs expressed by SCLCs. 3.1. x-Conotoxin GVIA x-Ctx GVIA was first shown by De Aizpurua et al. to antagonise KCl-induced influx of radioactive calcium in a SCLC cell line [28]. This critical finding was compromised by the interpretation that the reported inhibition was due to x-Ctx GVIA effects on L-type VDCC. The dominant view was, indeed, that x-Ctx GVIA was binding to both L- and N-type VDCCs in neurons. x-Ctx GVIA was later shown to bind with high specificity to N-type, but not L-type VDCCs in vertebrate neuronal cells [30]. In the light of these findings, the results by DeAizpurua et al. could be re-interpreted as an evidence of N-type VDCCs in SCLC cells. We studied x-Ctx GVIA effects on human SCLC cells in binding [29], Fura-2 [29], patch-clamp [31, 32] and release [31] experiments. 125I-Ctx GVIA binds with very high affinity to human SCLC both from cell lines and from fresh tumour biopsies. 125I-Ctx GVIA binding was antagonised by cold x-Ctx GVIA, neomycin and high calcium, similar to neurons, but was unaffected by DHPs or verapamil, L-type VDCCs ligands. Using both Fura-2 and the patch clamp technique we showed that x-Ctx GVIA was blocking a component of calcium increase [29], or calcium currents [31, 32], not blocked by DHPs, and vice versa. Therefore from these studies the simultaneous presence of L- and N-type VDCCs in SCLC cells was demonstrated. Interestingly, the co-application of saturating concentrations of DHPs and x-Ctx GVIA left a component of calcium increase [29], and calcium currents [31, 32], unaffected, suggesting the presence in SCLC cells of also a non-L, non-N VDCC component as previously reported in neurons (see below). KCl-induced depolarisation induces a cadmium-sensitive serotonin release from SCLC cells [31]. x-Ctx GVIA was found to block in a concentration-dependant manner the KClstimulated release of serotonin, demonstrating that N-type VDCCs are not only present in SCLC cells but that also play an important role in excitation-secretion coupling. x-Ctx GVIA was later shown to selectively block VDCCs encoded by the α1B VDCC subunit, and subsequently mRNA encoding for the α1B subunit was detected in SCLC cells [31].
Sher et al. 3.2. x-Agatoxin IVA x-Aga IVA is a peptide toxin isolated from the venom of the funnel web spider Agelenopsis aperta. It has been shown to bind and block at nanomolar concentrations non-L, non-N type VDCCs in cerebellar Purkinje neurons ([33] and references therein). At similar or slightly higher concentrations x-Aga IVA was later shown to block a number of non-L, non-N VDCCs in neurons and endocrine cells, these channels being now defined as P or Q or P/Q, depending on the cell types and/or the authors [33]. We found that x-Aga IVA blocked concentrationdependently KCl-induced serotonin release from SCLC cells [31] and the DHP- and x-Ctx GVIA-resistant calcium currents [31, 32]. Other groups [34, 35] reported similar findings on SCLC calcium currents. Our data on the role of these P/Q type VDCCs in excitation-secretion coupling were further confirmed by Viglione et al. [35] who showed that x-Aga IVA inhibited depolarisationinduced secretion (capacitance measurements) in single SCLC cells. Subsequently, P/Q-type VDCCs have been shown to be encoded by isoforms of the α1A gene [33], which we [31] and others [36] have shown to be expressed in the SCLC. 3.3. x-Conotoxin MVIIC x-Ctx MVIIC is another conotoxin with VDCC selectivity, isolated from the venom of the cone-snail Conus magus. It blocks with different affinity both N-, P- and Q-type VDCCs in both neurons and endocrine cells [37]. x-Ctx MVIIC was found to block calcium influx, calcium currents, and single cell secretion in SCLC cells [35, 38], supporting the evidence obtained with x-Aga IVA that P/Q-type VDCC are present in SCLC cells and participate in excitation-secretion coupling. At tracer concentrations and under conditions where binding to N-type VDCCs is prevented, 125I-x-Ctx MVIIC has been shown to bind selectively to the P/Q type VDCCs expressed by SCLC cells [38]. As mentioned above, α1A transcripts coding for P/Q-type VDCCs are present in SCLC cells. 4. Nicotinic and VCDD neurotoxins in normal and pathological lung What is the evidence that nAchRs and VDCCs, specifically those found in SCLC cells, are present in normal human lung? Very scant, indeed. Are they over-expressed in tumour cells but not in normal cells? Very little is known, but some evidence from other species is now emerging. 4.1. Neuronal nicotinic receptors ‘Neuronal’ nicotinic receptors have recently been found in several non-tumoural, non-neuronal, tissues. α7 recep-
Peptide neurotoxins tors have been found in developing muscle [39], in differentiating tendon and periosteum [40], and in lymphocytes [41]. Interestingly, the presence of α7 receptors was recently demonstrated in both cultured lung neuroendocrine cells [16, 42] and in normal lung cells of the squirrel monkey [43], where it is enriched in neuroendocrine and alveolar type II cells. α3 receptors have been found in lymphocytes [44], thymocytes [45] and in several ‘lining’ cell types such as endothelial cells [46], cheratinocytes [47], and most important in the context of this review, bronchial epithelial cells [48]. In all these cells types nicotinic receptor activation could be involved in calcium dependent processes, such as cell differentiation, and cytoskeletal re-arrangements related to cell mobility [47]. At least in some of the above tissues, the presence of the nicotinic receptors was also confirmed at the protein level and functionally in terms of ion channel activity. Tobacco or nicotine exposure has been shown to increase hormone release in the lung of hamsters and to cause hyperplasia of lung neuroendocrine cells in this species [49, 50]. However, in these studies, no evidence was found for direct nicotinic receptor activation or, more importantly, for a specific role of nicotinic receptor subtypes. In the context of the possible role of α7 in the genesis of SCLC, two recent findings are very important: α7 has been implicated in the in vitro proliferation of primary cultures of rabbit lung neuroendocrine cells [42], with results very similar to those previously found in human SCLC cells [14–16, 26]. Furthermore, in the squirrel monkey study mentioned above, it was also found that chronic exposure of pregnant monkeys to tobacco smoke caused both an up-regulation of α7 expression in the lung cells of the new-borns, and an enhanced proliferation of their neuroendocrine cells [43]. Nicotinic receptors [8, 9] α3 expression [48] and nicotinic-induced cell proliferation [8, 16] were also demonstrated for tumoural and normal non-endocrine lung cells in vitro. An interesting recent finding was that not only nicotine, but also nicotinederived nitrosamines such as NNK and NNN, which are potent carcinogenic compounds, bind specifically to nicotinic receptors in lung cells, with selectivity for α7 and α3, respectively [16]. We can summarise by saying that evidence is now emerging on the presence of ‘neuronal’ nicotinic receptors in normal lung cells and on the possible role of these receptor is the stimulation of cell proliferation. Similar findings in human lung epithelium and the direct demonstration of the link between nicotinic receptor activation in neuroendocrine cells and SCLC induction, are not yet available, but may be forthcoming. 4.2. Neuronal calcium channels Like nicotinic receptors, ‘neuronal’ calcium channels have also been found in a number of non-neuronal tissues.
931 Examples include specialised secretory cells such as adrenal chromaffin cells and tumours [30] pancreatic beta cells [37, 51], pituitary cells [30], and testicular Sertoli cells [52]. Very little information is available on the presence of these channels in normal or pathological pulmonary cells, besides human SCLC. Non-characterised calcium currents are indeed present in in vitro cultured, O2-sensitive, neuroendocrine cells of the rabbit lung [53], but no further information on the molecular nature of these receptors is available. Recently, Schuller et al. have reported x-Ctx GVIA and x-Aga IVA effects in primary cultures of pulmonary neuroendocriene cells, very similar to those previously published on human SCLC [42, 54]. In particular these toxins reduce calcium fluxes and hormone release also from these normal lung cells.
5. Nicotinic and VDCC neurotoxins and paraneoplastic disorders 5.1. Paraneoplastic disorders Paraneoplastic neurological syndromes, which can affect both the central and peripheral nervous system, are associated with an underlying cancer but not attributable to the physical presence of the tumour and/or its metastases. These syndromes are recognised in association with only a limited number of tumours, for example SCLC and certain gynaecological tumours. Peptide neurotoxins have been especially useful in diagnosing the presence of autoantibodies in peripheral paraneoplastic autoimmune disorders. As the neurological symptoms in these disorders often precede the detection of the tumour by several months, these diagnostic tests are particularly useful in the early detection of underlying neoplasms. More specifically, radioimmunoassays have been developed to detect anti-AChR antibodies in patients with myasthenia gravis using 125I-α-Bgtx in patients with and without an underlying thymoma. [55]. Acquired neuromyotonia (Isaac’s syndrome) is characterised by hyperexcitability of the muscle fibres resulting in continuous muscle activity or myokymia. Although in the majority of patients the aetiology of the disease is unknown, approximately 20% of patients have an associated SCLC or thymoma. Antibodies directed against voltage-dependent potassium channels have been implicated as causative agents in this disorder and may be detected using VDKCs extracted from human brain [56] and labelled with α-dendrotoxin, a toxin purified from the venom of the snake Dendroaspis angusticeps. Around 50% of patients present with these autoantibodies. Autoantibodies against neuronal AchR and VDCC have also been found in a number of different paraneoplastic disorders and these will be discussed below.
932 5.2. Nicotinic receptors Conflicting reports on the presence or absence [57, 58] of anti-α7 receptors autoantibodies, as detected by 125I-αBgtx radioimmunoassay, have been described in patients with classical myasthenia gravis, a disease without known SCLC association. In the context of SCLC, and the above described expression of α-Bgtx-sensitive, α7 ‘autoantigens’ in this tumour, it is intriguing that two out of seven LEMS patients tested in the work mentioned above [58], had anti-α7 autoantibodies able to immunoprecipitate 125 I-α-Bgtx-labelled receptors extracted from IMR32 human neuroblastoma cells. Furthermore both Balestra et al. [58] and Vernino et al. [59] found that a minority of LEMS patients also produce autoantibodies that recognise α3 nicotinic receptors extracted from SY5Y human neuroblastoma cells, similar to the α3 receptors expressed in SCLC cells (see above). Assay of a larger population of LEMS sera will be crucial in determining the real incidence of these α7 and α3 autoantibodies. 5.3. Calcium channels Calcium channels were first described in 1981 as a major triggering autoantigen expressed by SCLC cells ([60, 61] and references therein). These anti-VDCC autoantibodies were shown to cross-react with nerve terminal calcium channels, causing a widespread deficit in neurotransmitter release and leading to the typical LEMS symptoms [60, 61]. However, the molecular identity of the SCLC/LEMS autoantigen was identified only more recently, an event that also led to the development of clinically useful diagnostic radioimmunoassays. Again peptide neurotoxins were crucial in these efforts. In 1989, we first reported that LEMS antibodies were able to immunoprecipitate 125I-Ctx GVIA-labelled N-type VDCCs extracted from the human neuroblastoma cell line IMR32 [62]. 90% of this first group of patients was found to be positive. However, in a subsequent study on a larger population [63] we found 79% of LEMS patients to be positive in this assay. Interestingly, in the latter paper 30% of SCLC patients without neurological complications (a large incidence considering how common is SCLC with respect to LEMS) were positive in the assay, while patients with other lung tumours or non-tumoural lung diseases were negative. Shortly after, these data on LEMS patients were confirmed by Lennon and Lambert [64] and by Leys et al. [65]. In the work by Lennon and Lambert, 52% of LEMS patients were positive for N-type autoantibodies, while this percentage increased to 76% in LEMS patients with SCLC. In the work by Leys et al. 44% of patients were positive for N-type autoantibodies while this percentage increased to 61% in LEMS patients without SCLC. The reasons for these discrepancies are not clear, but are likely to be due to slight modifications in the immunoprecipitation protocols and in the heterogeneity of
Sher et al. the LEMS population investigated. In further publications, N-type autoantibody positive patients were found in percentages of 55% [66], 49% (73% in LEMS with SCLC) [38], 62% (75% in LEMS with SCLC) [67], and 58% [68]. A breakthrough in the field happened in 1995 when Motomura et al. [69] and Lennon et al. [38] reported an improved assay for LEMS autoantibodies which was based, again, on the use of a different peptide neurotoxin, x-Ctx MVIIC (see above). The rationale behind using this toxin instead of x-Ctx GVIA was based on the finding that x-Ctx MVII-sensitive P/Q-type VDCCs subserve neurotransmitter release at the mammalian neuromuscular junction [70, 71]. Although a major component in neurotransmitter release at the amphibian neuromuscular junction [72, 73], the x-Ctx GVIA-sensitive VDCC appears to be only a minor component in mammals. So the role of anti-P/Q-type VDCC appears to be central to LEMS. A larger percentage of LEMS patients are positive in the x-Ctx MVIIC-based assay than in the x-Ctx GVIA-based assay. The values reported were 85% (90% in LEMS with SCLC) in [74], 95% (100% in LEMS with SCLC) in [38], 74% (85% in LEMS with SCLC) in [67]. Using recombinant human VDCC subtypes expressed in HEK293 cells, Lang et al. [75] also showed that LEMS immunoglobulin (IgG) selectively depressed the calcium fluxes in HEK cells expressing α1A (P/Q-type) VDCCs, and not in HEK cells expressing α1B (N-type), α1D (L-type) or α1E (R-type) VDCCs. Furthermore, in a subsequent paper [76] the same IgGs depressed both P-type currents in cerebellar Purkinje cells and Q-type currents in cerebellar granule cells, suggesting, indeed, that both P- and Q-type VDCCs are encoded by the α1A gene. This finding was recently confirmed by molecular studies [33]. Since SCLC express both x-Ctx GVIA-sensitive N-type VDCCs and x-Ctx MVIIC-sensitive P/Q-type VDCCs, both of them could potentially trigger an autoimmune response. The autoimmune response to the P/Q-type VDCCs is likely to be the major component of the neuromuscular deficits in these patients because these channels are known to control acetylcholine release from the neuromuscular junction. What is known then about the autoimmune response to the N-type VDCC? One possibility is that anti N-type autoantibodies could affect neurotransmitter release in the autonomic nervous system where N-type VDCCs play an important role [77]. This could be responsible for the autonomic dysfunctions often found in LEMS patients. However, it has recently been shown [78] that P/Q-type VDCCs participate in neurotransmitter release from some neurons of the autonomic nervous system, implicating that P/Q-type autoantibodies could be relevant also for functional deficits at these sites [79, 80]. Another possibility is that antibodies directed against N-type VDCCs are not functionally active, as implied by the work of Lang and
Peptide neurotoxins colleagues [75, 76] but are immunoprecipitated only because of the heterogeneous population of autoantibodies recognising the intracellularly located VDCC beta subunits [81]. Anti beta subunit autoantibodies have been found, indeed, in a significant percentage of LEMS patients [82, 83]. Although these antibodies are not pathogenetic, they could still immunoprecipitate N-type VDCCs. Cross-immunoprecipitation via beta subunits is clearly occurring and can justify most of the immunoprecipitation data with x-Ctx GVIA-labelled N-type VDCCs. It is worth mentioning, however, that LEMS sera have been shown to down-regulate N-type VDCCs [34, 84–89] as well as other VDCC subtypes [34, 84–89] in intact living cells suggesting they might also recognise the N-type α1B and other subunits directly from extracellular epitopes. The discrepancy between these data and the lack of effect on recombinant α1B VDCC in HEK cells [75, 76], could be due to a difference between recombinant versus native N-type VDCCs or, again, to the heterogeneity of patients’ autoimmune responses. Anti-VGCC antibodies have also been investigated in patients with paraneoplastic cerebellar degeneration (PCD). This devastating paraneoplastic disorder is characterised by a subacute onset of cerebellar dysfunction with gait and limb ataxia, dysathria and sensory deficits. Pathologically the disease is characterised by loss of cerebellar Purkinje cells whilst many patients have a specific anti-neuronal antibody (anti-Hu). In a study of 57 patients with PCD anti-P/Q-type VGCC were detected in 20% of patients with this anti-neuronal antibodies, but also in 16% of patients without [90]. P/Q- or N-type VDCC autoantibodies have been found at low titers also in non LEMS neurological patients, as well in SCLC patients without LEMS (see above). In particular, these antibodies were found in 54% of patients with paraneoplastic neurological complications of lung, breast or ovarian cancer, in 24% of patients with cancer without neurological disorders, 23% of patients with sporadic ALS, but only in 3% myasthenia gravis or epileptic patients [38]. 6. Prospectives Analysing all the above reported research, it is quite striking how important were snake, spider and snail peptide neurotoxins in the clarification of the presence and in the evaluation of the functional role of ligand-gated and voltage-gated ion channels in human SCLC cells. Furthermore, as it happened 20 years before in the case of α-Bgtx and myasthenia gravis [1, 2], the use of x-Ctx GVIA and x-Ctx MVIIC dramatically improved our knowledge and our ability to diagnose the Lambert-Eaton myasthenic syndrome, a neurological disorder most of the times associated with SCLC.
933 Another conotoxin (x-Ctx MVIIA) from Conus magus, which selectively targets N-type VDCCs, as x-Ctx GVIA does, but in a reversible manner, has already entered the clinic, where it is being evaluated as an analgesic. With some imagination, and a large dose of optimism, we could also foresee a clinical usefulness for other peptide toxins, or perhaps drugs modelled on them, for the treatment of SCLC itself. New peptide toxins, or even some already available, should also be further used to study in more detail other ion channels not yet fully characterised in SCLC cells (Na+ channels, K+ channels, α3 nicotinic receptors etc.), their possible role in tumour growth and their possible involvement in other autoimmune neurological disorders.
Acknowledgments We would like to acknowledge the continuous support of many neurologists who over the years provided us with patient’s sera.
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