TiPS - August 2990 [Vol. 121
305
Understanding of the molecular basis of drug resistall _ 3 in cancer reveals new targets for chh _notherapy The goal of using multiple cytotoxic chemotherapeutic drugs to cure human cancers has been partially achieved for some hematological malignancies, testicular cancer and childhood cancers, but has remained elusive for the bulk of adult-onset metastatic cancers. A recent international conference* addressed this issue. One of the major problems in cancer therapy is the development of resistance to cytotoxic drugs. Gregory Curt (NCI, Bethesda) suggested that the development of drug resistance should be considered as a step in malignant progression along with uncontrolled proliferation, metastasis and dedifferentiation. The beststudied molecule associated with multidrug resistance - the ATPdependent efflux pump P-glycoprotein (P170) - confers resistance to many na:ural-product cytotoxic drugs including vincristine, vinblastine, actinomycin D, gramicidin, doxorubicin, colchicine, etoposide {VP-16) and teniposide (VM-26). P170-mediated cellular drug efflux can be prevented, and thus resistance can be reversed, by several drugs, including the Ca*+ antagonist verapamil (for review, see Refs 1 and 2; Fig. 1). However, preliminary data from tissue culture systems are now beginning to define additional mechanisms of drug resistance, such as that involving glutathione transferasme (see below). P-glycoprotein is encoded by genes tenmed MDRl in humans, m&la and mdrlb in mice (also known as mdr3 and mdrl, respectively), and pgpl and pgp2 in hamsters ‘(Piet Borst, Netherlands Cancer Institute). Each of these species also contains a closely related gene (the MDR2/3 gene in humans) whose function remains unknown. The mdr gene family is part of a superfamily encoding AT&dependent transport proteins, which consist of multiple
transmembrane domains (12 in the case of P170) and highly conserved ATP bind:ng and utilization sites (two for P170). This superfamily of membrane transporter: includes bacterial and protczoan members with diverse function,; such as secretion of peptide mating factors, polypeptide toxin secretion, chloroquine resistance, nutrient transport, and the recently discovered cystic fibrosis gene product CFTR (Borst). Igor Roninson (University of Illinois College of Medicine) described studies that are beginning to define structure-function relationships of I’170 in human KB carcinoma cells. Using sitedirected mutagenesis to inactivate either or both nucleotide binding sites reveals that both of these sites are essential for action of the multidrug transporter. In addition, a Gly+Val mutation of P-glycoprotein at amino acid residue 185 (in the first cytoplasmic loop, near the third transmembrane sequence) alters the specificity of the pump, apparently by affecting drug transport but not initial binding. P170 is not only found in tumor cells: it is produced normally in some tissues (particularly kidney, liver and colon) and at certain developmental stages. Thus, using a highly sensitive assay for MDRl RNA based on the polymerase chain reaction, expression of the MDRl gene has been detected in human cancers as a normal consequence of their differentiated transport function, as a characteristic associated with their malignant state, or as a characteristic acquired during cancer chemotherapy treatment (Roninson). Antonio Fojo (NCI, Bethesda) has also investigated transcription of the MDRl gene (using irl sit14 hybridization and RNase protection assays to detect the mRNA) in both normal and malignant states of human adrenal gland, kidney and colon tissue. In cancer cell lines from these tissues, DMF, DMSO and butyrate induce El70 as one aspect of differentiation. In neuroblastoma cells in culture, retinoic acid has the same effect.
These results reinforce the primary view that expression of p-glycoprotein in many cancers is a function of the state cf differentiation of the tumor. In addition polymorphic variations have been detected in MDRl genes expressed in several human cancer cell lines. Evidence is also emerging of differences in expression patterns and function of the rrzdrla and rndrlb gene products in rodents. The rndrla gene product can be detected using monoclonal antibodies in uterine endothelial cells early (days l-7) in pregnancy, but this form then disappears and mdrlb is expressed late in pregnancy in lumenal epithelial cells (Victor Ling, Ontario Cancer Institute). While verapamii is capable of reversing nrdrla- as well as mdrlb-mediated drug resistance, the rndrla gene product seems to be much more sensitive than the nzdrlb gene product to the steroid hormone progesterone as a novel reversing agent. In addition, the expression of mdrlb was observed in secretion glands of the mouse and rat endometrium - a hormonal target of progesterone. Interestingly, the promoter of the mdrlb gene contains a glucocorticoid response element (GRE; the consensus sequence for regulation by glucocorticoid and progesterone receptors), in contrast to mdria (Susan Horwitz, Albert Einstein College of Medicine). The observation that cultured hamster cells that survive two sequential X-ray doses also appear to contain high levels of P-glycoprotein provides a new link between X-ray resistance and drug resistance (Ling). Model systems A variety of systems have proved useful in the study of the cell and molecular biology of P170-mediated drug resistance. Michael Gottesman (Na:ional Cancer Institute, Bethesda) described a vesicle transport system in which ATP serves as the preferred energy source, and a system epithelial-derived of polarized, MDCK (canine kidney) cells that has allowed the demonstration that epikhelia in which P-glycoprotein i.. found on apical membranes transport drugs from their basal to their apical surfaces. This is a model for the transepithelial transport of drugs in the kidney,
TiPS - Alrglrst 2990 [Vol.
DR
Plasma Membrane
I
w
I
fig.
CYTOSOL
1. Mechenistn of action of P-gfycopmfein.Lett: P-g/ycoprolein uses energy of ATP to transport drug molecules Right: Vempamdinhibitstrampof?, by competing for the drug binding site. (Taken from Ref. 1.)
liver and intestine by P-glycoprotein. Photoaffinity labeling of doxorubicin-associated membrane proteins revealed that in multidrug-resistant cells P-glycoprotein acts like a vacuum cleaner, r-emoving essentie,ly all of the doxorubicin from the membrane. A new in uiuo model of drug resistance, based on transgenic mice expressing the human MDRl gene in their bone marrow, has also been created (Gottesman). The bone marrow is normally a major site of toxicity in chemotherapy. However, in this new model blood cell numbers are not reduced in response to chemotherapy unless the human P170 is inhibited by an agent that reverses drug resistance. Thus it may be useful for screening drugs to determine if bone marrow toxicity is limiting, and for screening of new agents for in viva reversal cf drug resistance. The microinjection of P170 mRNA into Xenoptls oocytes is now being exploited as an in vitro system to study the functions of proteins encoded by mdr genes, and to characterize agents that inhibit their action (Horwitz:. Reversing multidrug resistance Verapamil is now being used clinically to reverse drug resistance. Sydney D. Slamon (University of Arizona) reported on the use of verapamil to inhibit the P-glycoprotein pump in multiple myeloma and malignant lymphoma. In these patients, relapse after chemotherapy is associated with expression of P-glycoprotein (detected histochemically in up to 60% of cases, compared with 3% of untreated patients). Verapamil
and quinine were able to reverse drug resistance in multiple myeloma and malignant lymphoma cell lines in vitro. They were therefore used in patient therapies after relapse to try to overcome drug significant Despite resistance. which toxicity cardiovascular required monitoring in an intensive care unit, the addition of verapamil to vincristine-adriamyciw dexamethasone therapy of multiple myeloma and malignant lymphoma resulted in a significant number of remissions in P-glycoproteinpositive patients. While these clinical results are encouraging, the cardiovascular side-effects of verapamil remain a major concern. Takahishi Tsuruo (Cancer Chemotherapy Center and University of Tokyo) reported progress on developing drugs that inhibit P170 more specifically; the quinoline derivative MS073 and the dihydropyridine derivative AHC-52 are giving promising preliminary results. Alternative approaches are also being considered. For example, monoclonal antibodies have been used to study drug resistance in various cancers, including softtissue carcinomas in children (Ling) and leukemia and lymphoma cell lines and primary tumors (Masanor Shimoyama, National Cancer Center Hospital, Tokyo). The presence of P170 immunoreactivity is associated with poor response to chemotherapy and poor prognosis. There is some hope, however, that antibodies may form the basis of a new therapeutic approach: they can cause ovarian tumor cytolysis and lymphocyte/monocyte-mediated turI~r cell killing in vitro (TSUNO). Another possibility
out of cell.
would be to investigate the utility of inhibitors of protein kinase C (see below).
q
cl
0
New targets for chemotherapy In addition to discussing multidrug resistance and ways in which it can be overcome, various speakers considered progress that has been made in defining new targets for chemotherapeutic drugs on the basis of better understanding of the cell biology of cancer cells. 0 Protein kinase C. P170 is a substrate for phosphorylation by protein kinase C, and it may prove possible to exploit this fact therapeutically. Indeed, protein kinase C inhibitors do reduce cellular accumulation of chemotherapeutic drugs in vitro (Tsuruo). There is also the possibility that inhibition of protein kinase C during chemotherapy of breast cancer may help prevent malignant progression and cross-resistance to antihormonal agents (Robert Dickson, Georgetown University, Washington). Acquired resistance to adriamycin in human breast cancer cells in vitro is associated with increased MDRl gene expression, and with increased protein kinase C activity. In addition, downregulation of estrogen receptor and upregulation of epidermal growth factor (EGF) receptor numbers occurs, coupled with hortumorigenesis mone-dependent and increased basement membrane invasiveness. MDRl gene transfection experiments suggest that MD::1 expression is not the direct cause of these changes, but protein kinase C may mediate
307 some of them: activation of protein kinase C with phorbol esters downregulates estrogen receptor and increases transcription of EGF receptor. 6 Gl~fa~~io~e S-~a~5fer~se x (GSTn). Expression of the GSTn gene appears to be induced both in precancerous lesions and during acquisition of drug resistance (Masami Muramatsu. University of Tokyo). in the rodent, the promoter of the gene has two TPAresponsive elements, one of which appears to render the gene hypersensitive to regulation by c-jun proto-oncogene. Transfection of cells with the GSTn gene to assess function has yielded mixed results. In CHO cells, gene transfection is associated with cellular resistance to cisplatin and carboplatin, but not adriamycin. In contrast, in NIH3T3 cells, gene transfer is associated with adriamycin but not cisplatin resistance.
0 Topoisomerases. These enzymes catalyse the relaxation of supercoiled DNA during replication. Their mechanism of action involves transient breakage of DNA, rotation and rejoining. Type I enzymes transiently break only one strand of DNA while type II enzymes transiently break both strands. The intermediates in the reaction contain enzyme-tyroline-O-phosphat~DNA linkages. Active chemotherapeutic drugs prevent reforming of DNA ester linkages, leaving permanently broken DNA linked to enzyme. The drug camptothecin inhibits topoisomerase I; the drug amsacrine inhibits topoisomerase !I. Using yeast mutants in topoisomerase I as a model system, James C. Wang (Harvard University, Cambridge) has demonstrated a direct relationship between camptothecin resistance and topoisomerase I levels. Leroy J. Liu (Johns
ROBERT B. DICKSON AND MICHAEL
M. GOTTESMAN*
References 1 Gottesman, M. h4. and Pastan, 1. (1988) TWld5 Piiffr?llacur. Sd. 9. 54-58 2 Lam, J. S. and Bahnsbn. R. R. (1989) Trcrrds Pknmmcol. Sci. 10, 369-373 3 Vickers, I’. J., Dickson, R. B. and Cowan, K. H. (1988) Trolds Plmrwncol. Sci. 9, 443-445 TPA: 12-C)-tetradecanoylphorboi-13-acetate
NMDA receptors. The properties of the NMDA system are such that this approach may shed light on events underlying persistent pain.
the involvement of excitatory amino acids in nociception came from behavioural studies in which NMDA agonists including induced nociceptive effects and the competitive antagonist AP5 had analgesic effects9. Electrophysiological studies suggest that the NMDA receptor is preferentially involved in spinal polysynaptic pathways whereas monosynaptic inputs use non-NMDA, presumably AMPA receptors’.3*6.
EAA and pain Early neurochemical studies revealed an uneven distribution of glutamate in the spinal cord, with mrire being found in the sensory dorsal horn areas than elsewhere (see Ref. 6). The demonstration of glutamate uptake into small ganglion cells was another strong indicator of a transmitter role in fine afferent fibres7. More recent anatomical studies show that excitatory amino acids such as glutamate are present in C-fibre‘ processes and their terminals in the dorsal horn, and that glutamate coexists in most substanceP-containing C fibres”; stimulation of nociceptive afferent fibres in the periphery may thus result in the co-release of a number of peptides together with excitatory amino acids in the spinal cord. First experimental evidence for
Plasticity and NMDA There is now evidence for an involvement of NMDA receptor mechanisms in long-term neuronal changes and plasticity in the forebrain’“. The NMDA receptorchannel complex is uniquely both ligand and voltage gated. The resting block of the NMDA receptor channel by Mg2* which is removed by depolarization provides a mechanism by which neuronal activity can be switched from low to high levels when the block is relieved, and this has important implications for no& ceptive signalling. The NMDA system could provide a substrate fo,-central alterations in pain and provide one basis for the observations that clearly show that nociceptive systems are not fixed and immutable but capable of considerable plasticity”.
A cure for wind up: NMDA receptor antagonists as potential analgesics For many years the mediators of nociceptive transmission from peripheral afferent C nociceptive fibres have been presumed to be predominantly peptide in nature’,2. Peptides such as substance I’, CGRP and the neurokinins have been studied widely as potential nociceptive transmitters, but studies have been complicated by the existence of multiple receptor subtypes and hampered by the absence of selective antagonists. The availability of selective antagonists for the excitatory amino acid receptor subtypes and channels, recently reviewed in TZS3-6, has allowed the potential role of excitatory amino acids in nociceptive transmission - parcicularly in the spinal cord - to be studied. Two lines of evidence support such a role. Firstly, glutamate has been shown neurochemitally to be co-located with peptides in sensory nerves. Secondly, the established phenomenon of ‘wind up’ in nociceptive neurons appears to be associated with
Hopkins University) described ii] viva applications of camptothecin and its derivatives as potent in. hibitors of human colon cancer in nude mice. While cell killing by topoisomerase-l-directed agents appears to involve replication fork arrest and possible DNA breakage, topoisomerase-R-directed drugs inhibit both replication and transcription.
305
Understanding of the molecular basis of drug resistall _ 3 in cancer reveals new targets for chh _notherapy The goal of using multiple cytotoxic chemotherapeutic drugs to cure human cancers has been partially achieved for some hematological malignancies, testicular cancer and childhood cancers, but has remained elusive for the bulk of adult-onset metastatic cancers. A recent international conference* addressed this issue. One of the major problems in cancer therapy is the development of resistance to cytotoxic drugs. Gregory Curt (NCI, Bethesda) suggested that the development of drug resistance should be considered as a step in malignant progression along with uncontrolled proliferation, metastasis and dedifferentiation. The beststudied molecule associated with multidrug resistance - the ATPdependent efflux pump P-glycoprotein (P170) - confers resistance to many na:ural-product cytotoxic drugs including vincristine, vinblastine, actinomycin D, gramicidin, doxorubicin, colchicine, etoposide {VP-16) and teniposide (VM-26). P170-mediated cellular drug efflux can be prevented, and thus resistance can be reversed, by several drugs, including the Ca*+ antagonist verapamil (for review, see Refs 1 and 2; Fig. 1). However, preliminary data from tissue culture systems are now beginning to define additional mechanisms of drug resistance, such as that involving glutathione transferasme (see below). P-glycoprotein is encoded by genes tenmed MDRl in humans, m&la and mdrlb in mice (also known as mdr3 and mdrl, respectively), and pgpl and pgp2 in hamsters ‘(Piet Borst, Netherlands Cancer Institute). Each of these species also contains a closely related gene (the MDR2/3 gene in humans) whose function remains unknown. The mdr gene family is part of a superfamily encoding AT&dependent transport proteins, which consist of multiple
transmembrane domains (12 in the case of P170) and highly conserved ATP bind:ng and utilization sites (two for P170). This superfamily of membrane transporter: includes bacterial and protczoan members with diverse function,; such as secretion of peptide mating factors, polypeptide toxin secretion, chloroquine resistance, nutrient transport, and the recently discovered cystic fibrosis gene product CFTR (Borst). Igor Roninson (University of Illinois College of Medicine) described studies that are beginning to define structure-function relationships of I’170 in human KB carcinoma cells. Using sitedirected mutagenesis to inactivate either or both nucleotide binding sites reveals that both of these sites are essential for action of the multidrug transporter. In addition, a Gly+Val mutation of P-glycoprotein at amino acid residue 185 (in the first cytoplasmic loop, near the third transmembrane sequence) alters the specificity of the pump, apparently by affecting drug transport but not initial binding. P170 is not only found in tumor cells: it is produced normally in some tissues (particularly kidney, liver and colon) and at certain developmental stages. Thus, using a highly sensitive assay for MDRl RNA based on the polymerase chain reaction, expression of the MDRl gene has been detected in human cancers as a normal consequence of their differentiated transport function, as a characteristic associated with their malignant state, or as a characteristic acquired during cancer chemotherapy treatment (Roninson). Antonio Fojo (NCI, Bethesda) has also investigated transcription of the MDRl gene (using irl sit14 hybridization and RNase protection assays to detect the mRNA) in both normal and malignant states of human adrenal gland, kidney and colon tissue. In cancer cell lines from these tissues, DMF, DMSO and butyrate induce El70 as one aspect of differentiation. In neuroblastoma cells in culture, retinoic acid has the same effect.
These results reinforce the primary view that expression of p-glycoprotein in many cancers is a function of the state cf differentiation of the tumor. In addition polymorphic variations have been detected in MDRl genes expressed in several human cancer cell lines. Evidence is also emerging of differences in expression patterns and function of the rrzdrla and rndrlb gene products in rodents. The rndrla gene product can be detected using monoclonal antibodies in uterine endothelial cells early (days l-7) in pregnancy, but this form then disappears and mdrlb is expressed late in pregnancy in lumenal epithelial cells (Victor Ling, Ontario Cancer Institute). While verapamii is capable of reversing nrdrla- as well as mdrlb-mediated drug resistance, the rndrla gene product seems to be much more sensitive than the nzdrlb gene product to the steroid hormone progesterone as a novel reversing agent. In addition, the expression of mdrlb was observed in secretion glands of the mouse and rat endometrium - a hormonal target of progesterone. Interestingly, the promoter of the mdrlb gene contains a glucocorticoid response element (GRE; the consensus sequence for regulation by glucocorticoid and progesterone receptors), in contrast to mdria (Susan Horwitz, Albert Einstein College of Medicine). The observation that cultured hamster cells that survive two sequential X-ray doses also appear to contain high levels of P-glycoprotein provides a new link between X-ray resistance and drug resistance (Ling). Model systems A variety of systems have proved useful in the study of the cell and molecular biology of P170-mediated drug resistance. Michael Gottesman (Na:ional Cancer Institute, Bethesda) described a vesicle transport system in which ATP serves as the preferred energy source, and a system epithelial-derived of polarized, MDCK (canine kidney) cells that has allowed the demonstration that epikhelia in which P-glycoprotein i.. found on apical membranes transport drugs from their basal to their apical surfaces. This is a model for the transepithelial transport of drugs in the kidney,
TiPS - Alrglrst 2990 [Vol.
DR
Plasma Membrane
I
w
I
fig.
CYTOSOL
1. Mechenistn of action of P-gfycopmfein.Lett: P-g/ycoprolein uses energy of ATP to transport drug molecules Right: Vempamdinhibitstrampof?, by competing for the drug binding site. (Taken from Ref. 1.)
liver and intestine by P-glycoprotein. Photoaffinity labeling of doxorubicin-associated membrane proteins revealed that in multidrug-resistant cells P-glycoprotein acts like a vacuum cleaner, r-emoving essentie,ly all of the doxorubicin from the membrane. A new in uiuo model of drug resistance, based on transgenic mice expressing the human MDRl gene in their bone marrow, has also been created (Gottesman). The bone marrow is normally a major site of toxicity in chemotherapy. However, in this new model blood cell numbers are not reduced in response to chemotherapy unless the human P170 is inhibited by an agent that reverses drug resistance. Thus it may be useful for screening drugs to determine if bone marrow toxicity is limiting, and for screening of new agents for in viva reversal cf drug resistance. The microinjection of P170 mRNA into Xenoptls oocytes is now being exploited as an in vitro system to study the functions of proteins encoded by mdr genes, and to characterize agents that inhibit their action (Horwitz:. Reversing multidrug resistance Verapamil is now being used clinically to reverse drug resistance. Sydney D. Slamon (University of Arizona) reported on the use of verapamil to inhibit the P-glycoprotein pump in multiple myeloma and malignant lymphoma. In these patients, relapse after chemotherapy is associated with expression of P-glycoprotein (detected histochemically in up to 60% of cases, compared with 3% of untreated patients). Verapamil
and quinine were able to reverse drug resistance in multiple myeloma and malignant lymphoma cell lines in vitro. They were therefore used in patient therapies after relapse to try to overcome drug significant Despite resistance. which toxicity cardiovascular required monitoring in an intensive care unit, the addition of verapamil to vincristine-adriamyciw dexamethasone therapy of multiple myeloma and malignant lymphoma resulted in a significant number of remissions in P-glycoproteinpositive patients. While these clinical results are encouraging, the cardiovascular side-effects of verapamil remain a major concern. Takahishi Tsuruo (Cancer Chemotherapy Center and University of Tokyo) reported progress on developing drugs that inhibit P170 more specifically; the quinoline derivative MS073 and the dihydropyridine derivative AHC-52 are giving promising preliminary results. Alternative approaches are also being considered. For example, monoclonal antibodies have been used to study drug resistance in various cancers, including softtissue carcinomas in children (Ling) and leukemia and lymphoma cell lines and primary tumors (Masanor Shimoyama, National Cancer Center Hospital, Tokyo). The presence of P170 immunoreactivity is associated with poor response to chemotherapy and poor prognosis. There is some hope, however, that antibodies may form the basis of a new therapeutic approach: they can cause ovarian tumor cytolysis and lymphocyte/monocyte-mediated turI~r cell killing in vitro (TSUNO). Another possibility
out of cell.
would be to investigate the utility of inhibitors of protein kinase C (see below).
q
cl
0
New targets for chemotherapy In addition to discussing multidrug resistance and ways in which it can be overcome, various speakers considered progress that has been made in defining new targets for chemotherapeutic drugs on the basis of better understanding of the cell biology of cancer cells. 0 Protein kinase C. P170 is a substrate for phosphorylation by protein kinase C, and it may prove possible to exploit this fact therapeutically. Indeed, protein kinase C inhibitors do reduce cellular accumulation of chemotherapeutic drugs in vitro (Tsuruo). There is also the possibility that inhibition of protein kinase C during chemotherapy of breast cancer may help prevent malignant progression and cross-resistance to antihormonal agents (Robert Dickson, Georgetown University, Washington). Acquired resistance to adriamycin in human breast cancer cells in vitro is associated with increased MDRl gene expression, and with increased protein kinase C activity. In addition, downregulation of estrogen receptor and upregulation of epidermal growth factor (EGF) receptor numbers occurs, coupled with hortumorigenesis mone-dependent and increased basement membrane invasiveness. MDRl gene transfection experiments suggest that MD::1 expression is not the direct cause of these changes, but protein kinase C may mediate
307 some of them: activation of protein kinase C with phorbol esters downregulates estrogen receptor and increases transcription of EGF receptor. 6 Gl~fa~~io~e S-~a~5fer~se x (GSTn). Expression of the GSTn gene appears to be induced both in precancerous lesions and during acquisition of drug resistance (Masami Muramatsu. University of Tokyo). in the rodent, the promoter of the gene has two TPAresponsive elements, one of which appears to render the gene hypersensitive to regulation by c-jun proto-oncogene. Transfection of cells with the GSTn gene to assess function has yielded mixed results. In CHO cells, gene transfection is associated with cellular resistance to cisplatin and carboplatin, but not adriamycin. In contrast, in NIH3T3 cells, gene transfer is associated with adriamycin but not cisplatin resistance.
0 Topoisomerases. These enzymes catalyse the relaxation of supercoiled DNA during replication. Their mechanism of action involves transient breakage of DNA, rotation and rejoining. Type I enzymes transiently break only one strand of DNA while type II enzymes transiently break both strands. The intermediates in the reaction contain enzyme-tyroline-O-phosphat~DNA linkages. Active chemotherapeutic drugs prevent reforming of DNA ester linkages, leaving permanently broken DNA linked to enzyme. The drug camptothecin inhibits topoisomerase I; the drug amsacrine inhibits topoisomerase !I. Using yeast mutants in topoisomerase I as a model system, James C. Wang (Harvard University, Cambridge) has demonstrated a direct relationship between camptothecin resistance and topoisomerase I levels. Leroy J. Liu (Johns
ROBERT B. DICKSON AND MICHAEL
M. GOTTESMAN*
References 1 Gottesman, M. h4. and Pastan, 1. (1988) TWld5 Piiffr?llacur. Sd. 9. 54-58 2 Lam, J. S. and Bahnsbn. R. R. (1989) Trcrrds Pknmmcol. Sci. 10, 369-373 3 Vickers, I’. J., Dickson, R. B. and Cowan, K. H. (1988) Trolds Plmrwncol. Sci. 9, 443-445 TPA: 12-C)-tetradecanoylphorboi-13-acetate
NMDA receptors. The properties of the NMDA system are such that this approach may shed light on events underlying persistent pain.
the involvement of excitatory amino acids in nociception came from behavioural studies in which NMDA agonists including induced nociceptive effects and the competitive antagonist AP5 had analgesic effects9. Electrophysiological studies suggest that the NMDA receptor is preferentially involved in spinal polysynaptic pathways whereas monosynaptic inputs use non-NMDA, presumably AMPA receptors’.3*6.
EAA and pain Early neurochemical studies revealed an uneven distribution of glutamate in the spinal cord, with mrire being found in the sensory dorsal horn areas than elsewhere (see Ref. 6). The demonstration of glutamate uptake into small ganglion cells was another strong indicator of a transmitter role in fine afferent fibres7. More recent anatomical studies show that excitatory amino acids such as glutamate are present in C-fibre‘ processes and their terminals in the dorsal horn, and that glutamate coexists in most substanceP-containing C fibres”; stimulation of nociceptive afferent fibres in the periphery may thus result in the co-release of a number of peptides together with excitatory amino acids in the spinal cord. First experimental evidence for
Plasticity and NMDA There is now evidence for an involvement of NMDA receptor mechanisms in long-term neuronal changes and plasticity in the forebrain’“. The NMDA receptorchannel complex is uniquely both ligand and voltage gated. The resting block of the NMDA receptor channel by Mg2* which is removed by depolarization provides a mechanism by which neuronal activity can be switched from low to high levels when the block is relieved, and this has important implications for no& ceptive signalling. The NMDA system could provide a substrate fo,-central alterations in pain and provide one basis for the observations that clearly show that nociceptive systems are not fixed and immutable but capable of considerable plasticity”.
A cure for wind up: NMDA receptor antagonists as potential analgesics For many years the mediators of nociceptive transmission from peripheral afferent C nociceptive fibres have been presumed to be predominantly peptide in nature’,2. Peptides such as substance I’, CGRP and the neurokinins have been studied widely as potential nociceptive transmitters, but studies have been complicated by the existence of multiple receptor subtypes and hampered by the absence of selective antagonists. The availability of selective antagonists for the excitatory amino acid receptor subtypes and channels, recently reviewed in TZS3-6, has allowed the potential role of excitatory amino acids in nociceptive transmission - parcicularly in the spinal cord - to be studied. Two lines of evidence support such a role. Firstly, glutamate has been shown neurochemitally to be co-located with peptides in sensory nerves. Secondly, the established phenomenon of ‘wind up’ in nociceptive neurons appears to be associated with
Hopkins University) described ii] viva applications of camptothecin and its derivatives as potent in. hibitors of human colon cancer in nude mice. While cell killing by topoisomerase-l-directed agents appears to involve replication fork arrest and possible DNA breakage, topoisomerase-R-directed drugs inhibit both replication and transcription.
