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A natural products chemist looks at the bystander effect!
Mutation Research 597 (2006) 39–42
A natural products chemist looks at the bystander effect! David J. Newman ∗ Natural Products Branch, Developmental Therapeutics Program, NCI-Frederick, PO Box B. Frederick, MD 21702, USA Received 6 January 2005; accepted 8 April 2005 Available online 18 January 2006
Abstract The bystander effect is a process whereby the medium in which cells are irradiated at very low levels is presumed to contain a “low molecular weight” entity that will induce cell death and/or genetic changes in fresh mammalian cells when they are incubated in the medium. Following discussions at a recent meeting in McMaster University, suggestions are made in this paper as to the possibility for the “entity” to be nitric oxide and recommendations are given as to methods that will permit a systematic (bio)chemical search to be performed in order to identify the “activator” and prove/disprove the hypothesis. Published by Elsevier B.V. Keywords: Bystander effect; Nitric oxide
1. Introduction As described by Mothersill and Seymour [1,2], the bystander effect is the biological effect seen in distant cells following the irradiation by very low-dose ionizing radiation. This is presumed to be due to low molecular weight “signals” that are received by “bystander cells” either in the same piece of tissue, but un-irradiated, or by transfer of the medium in which the irradiated cells were exposed, to fresh cells that then undergo cell death or other untoward effects mediated by the medium and what ever it contains. At a recent workshop held at McMaster University (October 2004), a group of investigators in the field of bystander and adaptive effects gave a significant number of presentations that in the case of the bystander effect, demonstrated that the effect was very rapid in media, exhibited a significant and very rapid calcium flux and in the case of irradiated tissues, the effect was mediated ∗
Tel.: +1 301 846 5387; fax: +1 301 846 6178. E-mail address: [email protected].
0027-5107/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.mrfmmm.2005.04.026
in some manner via the tight “gap junctions”, since inhibition of gap junction transport stopped the effects in neighboring cells. 2. Commentary on the bystander effect (BE) as currently described The BE to some extent resembles the process known in microbiology as “quorum sensing”, whereby a single microbial cell will send out a chemical signal that varies from microbe to microbe in order to recruit other cells into a “quorum” that will then permit pathogenic deposition or in a number of cases cause phenotypic shifts in microbes. There have been some recent reviews of this field covering chemical agents, mechanisms and description of the process as a “signal transduction cascade” including kinase activation and inactivation and the interested reader should consult the following reviews [3–6] for specific details. From the data provided by Mothersill and Seymour, the “factor” appears to be of low molecular weight and one of the first manifestations of the response in their
40
D.J. Newman / Mutation Research 597 (2006) 39–42
“tester cells” is the production of a very rapid calcium flux. From basic biochemistry of the cell, a mechanism that can be activated and regulated by such a calcium flux and whose activation meets the criterion of (production of) a small molecular weight, highly diffusible chemical entity, is the activation of the NOS I and NOS III (E.C. 1.14.13.39) isozymes. These may be better known by their trivial names of n-NOS or neuronal nitric oxide synthase and e-NOS or endothelial nitric oxide synthase. There is a third isozyme, NOS II that is an inducible system, or iNOS, which could play a role, but although its activity is dependent upon bound calmodulin, its initiation is not calcium dependent. Often the first physiological target for NO is the activation of soluble guanyl cyclase [7], which then causes elevation of cGMP which can account for the subsequent physiological effects of elevated NO. Since NO is also a free radical, as it has an unpaired electron, it may also interact directly with a variety of cellular components including proteins, lipids and nucleic acids and any transition metals therein. There have been some recent reviews on the NOS isozymes [8], their activities and inhibitors [9,10], and most importantly, some idea as to the effects when these enzymes are “knocked out” in rodents [8] and also what types of small chemical entities can act as inhibitors of NO production by modulation of the isozyme(s) [8–10].
ii.
iii.
iv.
v.
3. The chemical perspective From the perspective of a chemist, and in particular, a chemist who was trained in comparative biochemistry and natural products, there were some significant “disconnects” amongst the presentations at the McMaster Meeting, frequently around the topics of “signals and subsequent effects”. In order to be able to decide whether the processes seen/described are due to a form of chemical signaling, it might be instructive to consider what is required from a chemical and biochemical perspective to be able to first identify the effect unambiguously (and here I am referring to the bystander effect as described by Mothersill and Seymour with media-borne reactions). In order to do this, there has to be agreement within the community on the following, so that comparative studies can be made both within laboratories and across continents: i. A decision as to which cell lines should be used to investigate the process, both tester strains (irradiated) and responder strains (bystanders). This should include a genetic analysis, knowledge of the number
vi.
of passages, decisions as to what range of passages should be used. The choice(s) of media. If at all possible, it would definitely be worth considering the use of defined media. In fact, if such a defined medium (no fetal calf serum or serum of any kind) could be used and the “bystander effect” is demonstrable, then it could make the chemist’s life a lot simpler. In the absence of such media, then the minimal serum level that will still show a response should be chosen. The method(s) of irradiation and the systems/levels involved should be standardized between investigators. The definition of what process should be used to transfer irradiated media from the irradiated cells to the responder cells. This may well involve filtration through sterile systems, and it should be realized that if any factor is proteinaceous, then one can have binding to certain filters. A Teflon 0.22 m filter in a Teflon carrier will minimize such binding. A definition for a particular system of what constitutes a response. This definition should be such that a formal definition of “unit of response” will be achievable. A simple example would be to use a dilution series of the irradiated medium and dilute until no response is seen in the tester strain. This, if a two-fold dilution, would be formally identical to the definition of a minimum inhibitory (in this case, activation) concentration as used in antibiotic assays. The reciprocal of the dilution would then give a definition of a “bystander unit”, which can be used to compare treatments, and most importantly, permit a chemist to see if they are producing any increase in purification as the unit/volume relationship will show this. This is a corollary to #v. If there is not a dilution series then the task of the chemist becomes much more difficult as the phenomenon may then be due to an activity “burst” that triggers a response but is not stoichiometric in nature. However, until one has a reproducible system to study, then such phenomena cannot be interpreted.
4. Nitric oxide involvement? Once a reproducible system is in place, then pharmacological intervention using some of the compounds shown in the reviews above, either substrate-based inhibitors which are usually variations on arginine, or a compound similar to those described by Vallance and Leiper [8] where there is a structural scaffold that provides an amidino, ureido or guanidine that will donate
D.J. Newman / Mutation Research 597 (2006) 39–42
41
Fig. 1. Inhibitors of the mammalian cell cycle. From [11]. Copyright Springer-Verlag 2003. Reprinted with permission.
hydrogen bonds to the glutamate residue at the active site of the NOS isozyme, plus a small hydrophobic group to provide further non-polar interactions in the active site and these would be linked to a grouping that gives isozyme selectivity. Alternatively, one might also be able to use variations on the cofactor, tetrahydrobiopterin, as described by Werner et al. [9] and Matter and Kotsonis [10] and references therein. If any of these compounds modulate the response in a defined system, then that could be taken as prima facie evidence of involvement of NO at the beginning of the BE. 5. Conclusion With the reproducible system established, giving similar results in different investigator’s hands, then pharmacological and molecular biological techniques can be coupled to chemistry in order to determine the sequence(s) of cellular events. However, in the absence of such a system, the field is doomed to continue in its current manner; viz, individual reports that are consistent within a group but cannot be (completely) confirmed in other laboratories. In order to demonstrate that relatively simple natural products will cause major modifications to the cell cycle
in mammalian cells, from inspection of the schematic in Fig. 1, which is reproduced by permission from a review by Meijer [11], one can see that a very large number of cell cycle processes can be inhibited by natural products that are not proteinaceous in nature. Thus one can easily envisage a simple molecule from a cell being the “initiator” of a cascade that is manifested as the BE. References [1] C. Mothersill, C. Seymour, Radiation-induced bystander effects and adaptive responses: the Ying and Yang of low dose radiobiology, Mutat. Res. 568 (2004) 121–128 (review). [2] C. Mothersill, C.B. Seymour, Radiation-induced bystander effects: implications for cancer, Nat. Rev. Cancer 4 (2004) 158–164 (review). [3] R. Daniels, J. Vanderleyden, J. Michiels, Quorum sensing and swarming migration in bacteria, FEMS Microbiol. Rev. 28 (2004) 261–289 (review). [4] K.M. Pappas, C.L. Weingart, S.C. Winans, Chemical communication in proteobacteria: biochemical and structural studies in signal synthetases and receptors required for intercellular signalling, Mol. Microbiol. 53 (2004) 755–769 (review). [5] L.-H. Zhang, Y.-H. Dong, Quorum sensing and signal interference: diverse implications, Mol. Microbiol. 53 (2004) 1563–1571 (review). [6] K.J. Hellingswerf, A network of net-workers: report of the Euresco conference on “Bacterial Neural Networks” held at San Feliu (Sain) from 8 to 14 May, Mol. Microbiol. 54 (2004) 2–13 (review).
42
D.J. Newman / Mutation Research 597 (2006) 39–42
[7] L.J. Ignarro, G. Cirino, A. Casini, C. Napoli, Nitric oxide as a signaling molecule in the vascular system: an overview, J. Cardiovasc. Pharmacol. 34 (1999). [8] P. Vallance, J. Leiper, Blocking NO synthesis: how where and why, Nat. Rev. Drug Discov. 1 (2002) 939–950 (review). [9] E.R. Werner, A.C.F. Gorren, R. Heller, G. Werner-Felmayer, B. Mayer, Tetrahydrobiopterin and nitric oxide: mechanistic and
pharmacological aspects, Exp. Biol. Med. 228 (2003) 1291– 1302. [10] H. Matter, P. Kotsonis, Biology and chemistry of the inhibition of nitric oxide synthases by pteridine-derivatives as therapeutic agents, Med. Res. Rev. 24 (2004) 662–684 (review). [11] L. Meijer, Le cycle de division cellulaire et sa regulation, Oncologie 5 (2003) 311–326 (review).
A natural products chemist looks at the bystander effect! David J. Newman ∗ Natural Products Branch, Developmental Therapeutics Program, NCI-Frederick, PO Box B. Frederick, MD 21702, USA Received 6 January 2005; accepted 8 April 2005 Available online 18 January 2006
Abstract The bystander effect is a process whereby the medium in which cells are irradiated at very low levels is presumed to contain a “low molecular weight” entity that will induce cell death and/or genetic changes in fresh mammalian cells when they are incubated in the medium. Following discussions at a recent meeting in McMaster University, suggestions are made in this paper as to the possibility for the “entity” to be nitric oxide and recommendations are given as to methods that will permit a systematic (bio)chemical search to be performed in order to identify the “activator” and prove/disprove the hypothesis. Published by Elsevier B.V. Keywords: Bystander effect; Nitric oxide
1. Introduction As described by Mothersill and Seymour [1,2], the bystander effect is the biological effect seen in distant cells following the irradiation by very low-dose ionizing radiation. This is presumed to be due to low molecular weight “signals” that are received by “bystander cells” either in the same piece of tissue, but un-irradiated, or by transfer of the medium in which the irradiated cells were exposed, to fresh cells that then undergo cell death or other untoward effects mediated by the medium and what ever it contains. At a recent workshop held at McMaster University (October 2004), a group of investigators in the field of bystander and adaptive effects gave a significant number of presentations that in the case of the bystander effect, demonstrated that the effect was very rapid in media, exhibited a significant and very rapid calcium flux and in the case of irradiated tissues, the effect was mediated ∗
Tel.: +1 301 846 5387; fax: +1 301 846 6178. E-mail address: [email protected].
0027-5107/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.mrfmmm.2005.04.026
in some manner via the tight “gap junctions”, since inhibition of gap junction transport stopped the effects in neighboring cells. 2. Commentary on the bystander effect (BE) as currently described The BE to some extent resembles the process known in microbiology as “quorum sensing”, whereby a single microbial cell will send out a chemical signal that varies from microbe to microbe in order to recruit other cells into a “quorum” that will then permit pathogenic deposition or in a number of cases cause phenotypic shifts in microbes. There have been some recent reviews of this field covering chemical agents, mechanisms and description of the process as a “signal transduction cascade” including kinase activation and inactivation and the interested reader should consult the following reviews [3–6] for specific details. From the data provided by Mothersill and Seymour, the “factor” appears to be of low molecular weight and one of the first manifestations of the response in their
40
D.J. Newman / Mutation Research 597 (2006) 39–42
“tester cells” is the production of a very rapid calcium flux. From basic biochemistry of the cell, a mechanism that can be activated and regulated by such a calcium flux and whose activation meets the criterion of (production of) a small molecular weight, highly diffusible chemical entity, is the activation of the NOS I and NOS III (E.C. 1.14.13.39) isozymes. These may be better known by their trivial names of n-NOS or neuronal nitric oxide synthase and e-NOS or endothelial nitric oxide synthase. There is a third isozyme, NOS II that is an inducible system, or iNOS, which could play a role, but although its activity is dependent upon bound calmodulin, its initiation is not calcium dependent. Often the first physiological target for NO is the activation of soluble guanyl cyclase [7], which then causes elevation of cGMP which can account for the subsequent physiological effects of elevated NO. Since NO is also a free radical, as it has an unpaired electron, it may also interact directly with a variety of cellular components including proteins, lipids and nucleic acids and any transition metals therein. There have been some recent reviews on the NOS isozymes [8], their activities and inhibitors [9,10], and most importantly, some idea as to the effects when these enzymes are “knocked out” in rodents [8] and also what types of small chemical entities can act as inhibitors of NO production by modulation of the isozyme(s) [8–10].
ii.
iii.
iv.
v.
3. The chemical perspective From the perspective of a chemist, and in particular, a chemist who was trained in comparative biochemistry and natural products, there were some significant “disconnects” amongst the presentations at the McMaster Meeting, frequently around the topics of “signals and subsequent effects”. In order to be able to decide whether the processes seen/described are due to a form of chemical signaling, it might be instructive to consider what is required from a chemical and biochemical perspective to be able to first identify the effect unambiguously (and here I am referring to the bystander effect as described by Mothersill and Seymour with media-borne reactions). In order to do this, there has to be agreement within the community on the following, so that comparative studies can be made both within laboratories and across continents: i. A decision as to which cell lines should be used to investigate the process, both tester strains (irradiated) and responder strains (bystanders). This should include a genetic analysis, knowledge of the number
vi.
of passages, decisions as to what range of passages should be used. The choice(s) of media. If at all possible, it would definitely be worth considering the use of defined media. In fact, if such a defined medium (no fetal calf serum or serum of any kind) could be used and the “bystander effect” is demonstrable, then it could make the chemist’s life a lot simpler. In the absence of such media, then the minimal serum level that will still show a response should be chosen. The method(s) of irradiation and the systems/levels involved should be standardized between investigators. The definition of what process should be used to transfer irradiated media from the irradiated cells to the responder cells. This may well involve filtration through sterile systems, and it should be realized that if any factor is proteinaceous, then one can have binding to certain filters. A Teflon 0.22 m filter in a Teflon carrier will minimize such binding. A definition for a particular system of what constitutes a response. This definition should be such that a formal definition of “unit of response” will be achievable. A simple example would be to use a dilution series of the irradiated medium and dilute until no response is seen in the tester strain. This, if a two-fold dilution, would be formally identical to the definition of a minimum inhibitory (in this case, activation) concentration as used in antibiotic assays. The reciprocal of the dilution would then give a definition of a “bystander unit”, which can be used to compare treatments, and most importantly, permit a chemist to see if they are producing any increase in purification as the unit/volume relationship will show this. This is a corollary to #v. If there is not a dilution series then the task of the chemist becomes much more difficult as the phenomenon may then be due to an activity “burst” that triggers a response but is not stoichiometric in nature. However, until one has a reproducible system to study, then such phenomena cannot be interpreted.
4. Nitric oxide involvement? Once a reproducible system is in place, then pharmacological intervention using some of the compounds shown in the reviews above, either substrate-based inhibitors which are usually variations on arginine, or a compound similar to those described by Vallance and Leiper [8] where there is a structural scaffold that provides an amidino, ureido or guanidine that will donate
D.J. Newman / Mutation Research 597 (2006) 39–42
41
Fig. 1. Inhibitors of the mammalian cell cycle. From [11]. Copyright Springer-Verlag 2003. Reprinted with permission.
hydrogen bonds to the glutamate residue at the active site of the NOS isozyme, plus a small hydrophobic group to provide further non-polar interactions in the active site and these would be linked to a grouping that gives isozyme selectivity. Alternatively, one might also be able to use variations on the cofactor, tetrahydrobiopterin, as described by Werner et al. [9] and Matter and Kotsonis [10] and references therein. If any of these compounds modulate the response in a defined system, then that could be taken as prima facie evidence of involvement of NO at the beginning of the BE. 5. Conclusion With the reproducible system established, giving similar results in different investigator’s hands, then pharmacological and molecular biological techniques can be coupled to chemistry in order to determine the sequence(s) of cellular events. However, in the absence of such a system, the field is doomed to continue in its current manner; viz, individual reports that are consistent within a group but cannot be (completely) confirmed in other laboratories. In order to demonstrate that relatively simple natural products will cause major modifications to the cell cycle
in mammalian cells, from inspection of the schematic in Fig. 1, which is reproduced by permission from a review by Meijer [11], one can see that a very large number of cell cycle processes can be inhibited by natural products that are not proteinaceous in nature. Thus one can easily envisage a simple molecule from a cell being the “initiator” of a cascade that is manifested as the BE. References [1] C. Mothersill, C. Seymour, Radiation-induced bystander effects and adaptive responses: the Ying and Yang of low dose radiobiology, Mutat. Res. 568 (2004) 121–128 (review). [2] C. Mothersill, C.B. Seymour, Radiation-induced bystander effects: implications for cancer, Nat. Rev. Cancer 4 (2004) 158–164 (review). [3] R. Daniels, J. Vanderleyden, J. Michiels, Quorum sensing and swarming migration in bacteria, FEMS Microbiol. Rev. 28 (2004) 261–289 (review). [4] K.M. Pappas, C.L. Weingart, S.C. Winans, Chemical communication in proteobacteria: biochemical and structural studies in signal synthetases and receptors required for intercellular signalling, Mol. Microbiol. 53 (2004) 755–769 (review). [5] L.-H. Zhang, Y.-H. Dong, Quorum sensing and signal interference: diverse implications, Mol. Microbiol. 53 (2004) 1563–1571 (review). [6] K.J. Hellingswerf, A network of net-workers: report of the Euresco conference on “Bacterial Neural Networks” held at San Feliu (Sain) from 8 to 14 May, Mol. Microbiol. 54 (2004) 2–13 (review).
42
D.J. Newman / Mutation Research 597 (2006) 39–42
[7] L.J. Ignarro, G. Cirino, A. Casini, C. Napoli, Nitric oxide as a signaling molecule in the vascular system: an overview, J. Cardiovasc. Pharmacol. 34 (1999). [8] P. Vallance, J. Leiper, Blocking NO synthesis: how where and why, Nat. Rev. Drug Discov. 1 (2002) 939–950 (review). [9] E.R. Werner, A.C.F. Gorren, R. Heller, G. Werner-Felmayer, B. Mayer, Tetrahydrobiopterin and nitric oxide: mechanistic and
pharmacological aspects, Exp. Biol. Med. 228 (2003) 1291– 1302. [10] H. Matter, P. Kotsonis, Biology and chemistry of the inhibition of nitric oxide synthases by pteridine-derivatives as therapeutic agents, Med. Res. Rev. 24 (2004) 662–684 (review). [11] L. Meijer, Le cycle de division cellulaire et sa regulation, Oncologie 5 (2003) 311–326 (review).