J Cancer Res Clin Oncol (1992) 118:238-239
C~6eer ~esearch Clinical 9 9 Springer-Verlag1992
Meeting report Membrane transport in multidrug resistance, development, and disease AACR Special Conference in Cancer Research* B. Jandrig and V. Wunderlich Central Institute for Cancer Research, Lindenberger Weg 80, O-1115 Berlin, Federal Republic of Germany Received29 September 1991/Accepted17 October 1991
Summary. The main goal of this meeting was to provide the scientists and clinicians active in this field with a comprehensive overview of the progress that has been made. The meeting was a forum in which new advances in membrane transport were discussed in depth and which gave new impulses for clinical applied research. Again, the importance of intensive cooperation between basic research and clinical use became evident during this symposium.
Introduction The importance of membrane transport phenomena was the subject of an outstanding meeting held in Banff, Canada, 10-I 3 March 1991. The meeting attracted about 250 participants from 12 countries. Different aspects of membrane transport were discussed in individual sessions. Multidrug resistance, chemomodulation of drug resistance, P-glycoprotein homologues and development, metabolite and drug transport in bacteria and eukaryotes, the cystic fibrosis gene and its product, phosphorylation and signal transduction were talked about. Presentations were made by more than 25 invited speakers and about 100 posters were displayed. The aim of the meeting was to improve the exchange of ideas and cooperation between different disciplines, especially between basic research in membrane transport and clinical approaches in cancer and cystic fibrosis.
Multidrug resistance The phenomenon of resistance to a broad variety of natural organic compounds that do not share any functional or structural similarities is called multidrug resistance (MDR). About 15 years ago V. Ling (one of the chairpersons of the meeting) and colleagues discovered P glyco* Co-sponsored by the National Cancer Institute of Canada Offprint requests to: V.Wunderlich
protein as a 170-kDa plasma membrane protein, which is now known to be encoded by a multidrug-resistance gene and to be sufficient to produce M D R in cancer cells. Pglycoprotein consists of two symmetrical halves, each has a set of six hydrophobic transmembrane regions and a putative ATP-binding domain, and functions as a pleiotropic transport protein that pumps quite different drugs out of cells. Highlights of the meeting were the lectures by I. Roninson (Chicago) and M. Gottesman (Bethesda). I. Roninson stressed that both nucleotide-binding sites are necessary for P glycoprotein function. By creating point mutations and by [3H]azidopine binding experiments he demonstrated a special function of the N-halfATPase for drug binding and of the C-half ATPase for pushing out the drug. Furthermore he presented new data about the role of P-glycoprotein in haematopoitic system stem cells. The most primitive progenitors are those stem cells with the highest CD34 level, the lowest rhodamine-123 dye uptake, and high P glycoprotein expression. Differentiated bone marrow cells are sensitive to chemotherapy. After upset of chemotherapy, patients recover, which means that the stem cells with high P glycoprotein expression survived. Not everyone interpreted the data in the same way. A high P-glycoprotein level is a good explanation for the low rhodamine uptake but the physiological function is still speculative. M. Gottesman outlined his model of P glycoprotein as a "hydrophobic vacuum cleaner", which is looking around the membrane for hydrophobic substances. The presented data strongly support his hypothesis and emphasize the importance of a lipid environment for transport, so that in different species different specificities are to be suggested. Using expression vectors for testing mdrl ATP-site chimeras only constructs with N- and C-half ATP-binding domains in the correct order generated drug resistance, whereas constructs with the C- and C-, N- and N-, and C- and N-half ATP-binding domains failed. K. Ueda (Kyoto) used mdrl-lacZ fusion genes to establish an assay system to measure the ATPase activity of P glycoprotein. Although the N-half-/%galactosidase
239 fusion protein showed ATPase activity no M D R phenotype could be seen. Experiments with C-half-fi-galactosidase fusion protein and the combination of both separate halves are under investigation. Further insight into the physiological function of P glycoprotein probably will come by inactivation of mdr genes by homologous recombination. J. Smit from P. Borst' group (Amsterdam) presented data on successful homologous recombination events of mouse mdrl as well as mdr2 genes in ES cells. Plans were announced to test these for their capability of contributing to chimeric mice and transfer of the inactivated mdr genes to the germ line.
Chemomodulation of drug resistance The main problem for the chemotherapy of cancer is the development of resistance. Current research is seeking new concepts of cancer treatment, based upon a more profound understanding of multidrug resistance. The great interest of the pharmaceutical industry in this field was documented by participants from about 20 companies. Reversal of drug resistance in in vitro and in vivo systems was obtained by verapamil, dipyridamol, probenecid, cyclosporin A, compazine, megestrole acetate and some others. M. Hitselberger (Indianapolis) used monoclonal-antibody-Vinca-alkaloid immunoconjugates to obtain regression of tumour xenografts. M. Gottesman discussed mdrl-transgenic mice as a rapid and reliable system to determine the bioactivity of agents that reverse multidrug resistance in animals. B. Chabner (Bethesda) spoke about clinical trials of reversal of multidrug resistance in non-Hodgkin's lymphoma using the Ca-channel blocker verapamil. It was pointed out that clinical studies concerning the use of reversing agents are still in the stage of pilot studies. Definitive and confirmatory studies are announced for the future. H. Chan (Toronto) reported her updated experiences in the relevance of P glycoprotein expression in childhood soft-tissue sarcoma. A sophisticated immunohistochemical technique with two different antibodies (C219, C494) was used to detect P glycoprotein in individual cells. About 90% of P-glycoprotein-negative sarcomas remain relapse-free, as compared to 0% of P-glycoprotein-positive sarcomas. Similar results were obtained in neuroblastoma and osteosarcoma. Consequently, P glycoprotein expression appears to be the single most important mechanism of resistance and the limiting factor to success.
Metabolite and drug transport in bacteria and eukaryotes A series of presentations characterized members of the mdr multigene family from other species e.g. Drosophila
(J. Croop, Boston), Caenorhabditis elegans (P. Borst, Amsterdam), and Plasmodium falciparum (A. Cowman, Melbourne). For different reasons these studies are quite important. First, the data extend ideas about the phylogenetie tree of and evolutionary relationship between members of the mdr family. Second, experimental model systems could become developed to provide new ways to investigate mechanisms of mdr regulation and function rigorously. Third, as in malaria, resistance of microorganisms against drugs is a growing problem of medicine in general. Understanding the basic mechanisms of drug resistance at the molecular and functional level should give hope for better approaches to therapy. A number of talks and posters dealt with metabolite transport systems that have great molecular and structural homology to the mdr family system. A special ATPdependent transport system for glutathione S-conjugates was described by T. Ishikawa (Heidelberg). A former assumption that glutathione derivatives could be physiological substrates of P glycoprotein did not get any support. Presentations about lactose permease, glucose transport proteins, Na+/Ca 2+ transporter, Na+/H + antiporter, and tetracycline efflux pumps opened up the whole problem of import and export of substances through cell membranes, which seem to be based on some similar mechanisms.
The cystic fibrosis gene and its product A major topic of this meeting represented the cystic fibrosis gene and its product, a transmembrane conductance regulator (CFTR). Cystic fibrosis is an epithelial disease characterized by abnormalities in water and electrolyte transport that lead to pancreatic and pulmonary insufficiency. The gene for CFTR shows structural similarities to the mdr gene family. Mutations in the CFTR gene result in failure of an epithelial cell chloride channel to respond to cAMP thereby imbalancing ion and fluid transport. L.-P. Tsui (Toronto) gave an analysis of mutations within the CFTR gene. Although 75 different types of mutations were detected, a special predominant threebase-pair deletion (A F5o8) in the ATP-binding domain is present on 70% of all cystic fibrosis chromosomes but never on normal chromosomes. A correlation between AFso 8 on no, one, or two alleles and pancreatic insufficiency was well in line with expected values. CFTR acts like a cAMP-regulated membrane chloride channel. This conclusion was drawn from data presented by N. Kartner (Toronto). A cloned CFTR eDNA was transferred into non-epithelial insect cells. Expression of CFTR endowed the cells with plasma membrane chloride conductance that can be activated by cAMP, but what is not yet clear ist how a defect in a chloride channel can lead to such diverse abnormalities of the cystic fibrosis phenotype.
C~6eer ~esearch Clinical 9 9 Springer-Verlag1992
Meeting report Membrane transport in multidrug resistance, development, and disease AACR Special Conference in Cancer Research* B. Jandrig and V. Wunderlich Central Institute for Cancer Research, Lindenberger Weg 80, O-1115 Berlin, Federal Republic of Germany Received29 September 1991/Accepted17 October 1991
Summary. The main goal of this meeting was to provide the scientists and clinicians active in this field with a comprehensive overview of the progress that has been made. The meeting was a forum in which new advances in membrane transport were discussed in depth and which gave new impulses for clinical applied research. Again, the importance of intensive cooperation between basic research and clinical use became evident during this symposium.
Introduction The importance of membrane transport phenomena was the subject of an outstanding meeting held in Banff, Canada, 10-I 3 March 1991. The meeting attracted about 250 participants from 12 countries. Different aspects of membrane transport were discussed in individual sessions. Multidrug resistance, chemomodulation of drug resistance, P-glycoprotein homologues and development, metabolite and drug transport in bacteria and eukaryotes, the cystic fibrosis gene and its product, phosphorylation and signal transduction were talked about. Presentations were made by more than 25 invited speakers and about 100 posters were displayed. The aim of the meeting was to improve the exchange of ideas and cooperation between different disciplines, especially between basic research in membrane transport and clinical approaches in cancer and cystic fibrosis.
Multidrug resistance The phenomenon of resistance to a broad variety of natural organic compounds that do not share any functional or structural similarities is called multidrug resistance (MDR). About 15 years ago V. Ling (one of the chairpersons of the meeting) and colleagues discovered P glyco* Co-sponsored by the National Cancer Institute of Canada Offprint requests to: V.Wunderlich
protein as a 170-kDa plasma membrane protein, which is now known to be encoded by a multidrug-resistance gene and to be sufficient to produce M D R in cancer cells. Pglycoprotein consists of two symmetrical halves, each has a set of six hydrophobic transmembrane regions and a putative ATP-binding domain, and functions as a pleiotropic transport protein that pumps quite different drugs out of cells. Highlights of the meeting were the lectures by I. Roninson (Chicago) and M. Gottesman (Bethesda). I. Roninson stressed that both nucleotide-binding sites are necessary for P glycoprotein function. By creating point mutations and by [3H]azidopine binding experiments he demonstrated a special function of the N-halfATPase for drug binding and of the C-half ATPase for pushing out the drug. Furthermore he presented new data about the role of P-glycoprotein in haematopoitic system stem cells. The most primitive progenitors are those stem cells with the highest CD34 level, the lowest rhodamine-123 dye uptake, and high P glycoprotein expression. Differentiated bone marrow cells are sensitive to chemotherapy. After upset of chemotherapy, patients recover, which means that the stem cells with high P glycoprotein expression survived. Not everyone interpreted the data in the same way. A high P-glycoprotein level is a good explanation for the low rhodamine uptake but the physiological function is still speculative. M. Gottesman outlined his model of P glycoprotein as a "hydrophobic vacuum cleaner", which is looking around the membrane for hydrophobic substances. The presented data strongly support his hypothesis and emphasize the importance of a lipid environment for transport, so that in different species different specificities are to be suggested. Using expression vectors for testing mdrl ATP-site chimeras only constructs with N- and C-half ATP-binding domains in the correct order generated drug resistance, whereas constructs with the C- and C-, N- and N-, and C- and N-half ATP-binding domains failed. K. Ueda (Kyoto) used mdrl-lacZ fusion genes to establish an assay system to measure the ATPase activity of P glycoprotein. Although the N-half-/%galactosidase
239 fusion protein showed ATPase activity no M D R phenotype could be seen. Experiments with C-half-fi-galactosidase fusion protein and the combination of both separate halves are under investigation. Further insight into the physiological function of P glycoprotein probably will come by inactivation of mdr genes by homologous recombination. J. Smit from P. Borst' group (Amsterdam) presented data on successful homologous recombination events of mouse mdrl as well as mdr2 genes in ES cells. Plans were announced to test these for their capability of contributing to chimeric mice and transfer of the inactivated mdr genes to the germ line.
Chemomodulation of drug resistance The main problem for the chemotherapy of cancer is the development of resistance. Current research is seeking new concepts of cancer treatment, based upon a more profound understanding of multidrug resistance. The great interest of the pharmaceutical industry in this field was documented by participants from about 20 companies. Reversal of drug resistance in in vitro and in vivo systems was obtained by verapamil, dipyridamol, probenecid, cyclosporin A, compazine, megestrole acetate and some others. M. Hitselberger (Indianapolis) used monoclonal-antibody-Vinca-alkaloid immunoconjugates to obtain regression of tumour xenografts. M. Gottesman discussed mdrl-transgenic mice as a rapid and reliable system to determine the bioactivity of agents that reverse multidrug resistance in animals. B. Chabner (Bethesda) spoke about clinical trials of reversal of multidrug resistance in non-Hodgkin's lymphoma using the Ca-channel blocker verapamil. It was pointed out that clinical studies concerning the use of reversing agents are still in the stage of pilot studies. Definitive and confirmatory studies are announced for the future. H. Chan (Toronto) reported her updated experiences in the relevance of P glycoprotein expression in childhood soft-tissue sarcoma. A sophisticated immunohistochemical technique with two different antibodies (C219, C494) was used to detect P glycoprotein in individual cells. About 90% of P-glycoprotein-negative sarcomas remain relapse-free, as compared to 0% of P-glycoprotein-positive sarcomas. Similar results were obtained in neuroblastoma and osteosarcoma. Consequently, P glycoprotein expression appears to be the single most important mechanism of resistance and the limiting factor to success.
Metabolite and drug transport in bacteria and eukaryotes A series of presentations characterized members of the mdr multigene family from other species e.g. Drosophila
(J. Croop, Boston), Caenorhabditis elegans (P. Borst, Amsterdam), and Plasmodium falciparum (A. Cowman, Melbourne). For different reasons these studies are quite important. First, the data extend ideas about the phylogenetie tree of and evolutionary relationship between members of the mdr family. Second, experimental model systems could become developed to provide new ways to investigate mechanisms of mdr regulation and function rigorously. Third, as in malaria, resistance of microorganisms against drugs is a growing problem of medicine in general. Understanding the basic mechanisms of drug resistance at the molecular and functional level should give hope for better approaches to therapy. A number of talks and posters dealt with metabolite transport systems that have great molecular and structural homology to the mdr family system. A special ATPdependent transport system for glutathione S-conjugates was described by T. Ishikawa (Heidelberg). A former assumption that glutathione derivatives could be physiological substrates of P glycoprotein did not get any support. Presentations about lactose permease, glucose transport proteins, Na+/Ca 2+ transporter, Na+/H + antiporter, and tetracycline efflux pumps opened up the whole problem of import and export of substances through cell membranes, which seem to be based on some similar mechanisms.
The cystic fibrosis gene and its product A major topic of this meeting represented the cystic fibrosis gene and its product, a transmembrane conductance regulator (CFTR). Cystic fibrosis is an epithelial disease characterized by abnormalities in water and electrolyte transport that lead to pancreatic and pulmonary insufficiency. The gene for CFTR shows structural similarities to the mdr gene family. Mutations in the CFTR gene result in failure of an epithelial cell chloride channel to respond to cAMP thereby imbalancing ion and fluid transport. L.-P. Tsui (Toronto) gave an analysis of mutations within the CFTR gene. Although 75 different types of mutations were detected, a special predominant threebase-pair deletion (A F5o8) in the ATP-binding domain is present on 70% of all cystic fibrosis chromosomes but never on normal chromosomes. A correlation between AFso 8 on no, one, or two alleles and pancreatic insufficiency was well in line with expected values. CFTR acts like a cAMP-regulated membrane chloride channel. This conclusion was drawn from data presented by N. Kartner (Toronto). A cloned CFTR eDNA was transferred into non-epithelial insect cells. Expression of CFTR endowed the cells with plasma membrane chloride conductance that can be activated by cAMP, but what is not yet clear ist how a defect in a chloride channel can lead to such diverse abnormalities of the cystic fibrosis phenotype.
