Med Oncol. & Tumor Pharmacother. Vol 9, No. 1, pp. 21-24, 1992
GENETIC
ASPECTS OF MULTIDRUG
0736-0118/90 $3.00 + .00
RESISTANCE
M A R C P A U L Y , *t F E R N A N D RIES 2 and M A R I O D I C A T O 2 ILaboratoire de recherche sur le cancer et les maladies du sang, Z.1. Grasbusch, L-3370 Leudelange, Grand-Duchy of Luxembourg 2Centre hospitalier de Luxembourg, Ddpartement d'Hemato-Oncologie, Rue Barbld 4, L-1210 Luxembourg (First submitted 1 November 1991." accepted 27 November 1991)
Gene amplification is responsible both for dihydrofolate reductase induced methotrexate resistance, and for the P-glyeoprotein encoding multigene family induced multidrug resistance. The 6 pairs of hydrophobic regions of the P-glycoprotein fold up in a snake-like structure through the lipidic layers of the cell membrane. Other detoxification mechanisms include the glutathione S-transferase 'pi', but without gene amplification. K ~ Words." Muitidrug resistance
Seven years ago, it was shown that the development of cellular resistance to one or several drugs involves the amplification of at least one specific gene) In cultivated cells treated by a low dose of an antimitotic substance, the amplification is achieved by the transiently blocked replication machinery of the cell, producing extra copies of many genes in such circumstances. The maintenance of the drug in the culture media creates a selection pressure which sorts out cells with several copies of one or a few genes allowing to develop resistance against the drug and to survive. For instance, a treatment of cultivated mouse cells with a low dosis of methotrexate (MTX), an inhibitor of the enzyme dihydrofolate reductase (DHFR), can induce a specific amplification of the corresponding DHFR gene (1 and references therein). The copy number of this gene increases with the MTX dosis, as an effect of selection by MTX. Resistance against MTX is then caused by an overproduction of the D H F R enzyme.
cells selected by colchicin and overexpressing this P-glycoprotein. The isolation of positive clones was achieved using a specific monoclonal antibody raised against the P-glycoprotein, in an immunobtot assay. In a further step, a specific cDNA probe of the P-glycoprotein gene was used in Northern blot assays to demonstrate the presence, in resistant cells, of a proportionally increased amount of specific mRNA of about 4.7 Kb. In Southern blot assays performed by Riordan and his coworkers on genomic DNA, the identification of eight specific bands showed the existence of several homologous genes already in nonresistant cells. These bands become even more pronounced in the case of resistant cells. They therefore suggested that these genes probably belong to a P-glycoprotein encoding multigene family, members of which are amplified during the appearance of the multidrug resistance phenotype. In resistant cells, up to 60 copies of each gene could be present.
Multidrug resistant mammalian cells overproduce a glycoprotein of a molecular weight of 170 Kd, after amplification of the corresponding geneY After synthesis, the P-glycoprotein is embedded in the plasma membrane of the cell.
Independently of each other, different laboratories showed that a homologous multigene family also exists in different normal or tumoral cells of other mammals (mouse, rat, man). 3'4 The genes of these families are similarly organized and this fact suggests that they have been well conserved among the different mammalian species, during evolution. Therefore, Kartner and Ling proposed that this P-gtycoprotein gene supra-family was probably generated in two steps: an internal duplication event of a single ancestral gene leads to the formation of a first ancient multigene family. Next, divergence from this basic family occurred at the dawn
The P-glycoprotein encoding part of a cellular gene was isolated by Riordan and his coworkers from a chinese hamster cDNA library, a3 This library was established in the bacteriophage expression vector .;tgtll using mRNAs from resistant chinese hamster ovary 'To whom correspondanceshould be addressed. 21
22 M. Pauly et al. of mammalian appearance. This divergence would then explain the slight structural differences observed among the P-glycoproteins produced in the cells of the different mammalian species. After isolation and cloning of the corresponding gene, the murine and human P-glycoprotein could be obtained in high amounts and sequenced. 5'6 The primary structure of these proteins shows a sequence of about 1,280 amino acids. A hydropathy analysis of the different regions of the proteins allowed to conclude that they are duplicated and contain six pairs of hydrophobic regions. This observation lead to the suggestion that these regions fold up in a snake-like structure and pass through the double lipidic layers of the cellular membrane. Some hydrophilic regions would be located towards the inner and outer cellular compartments and bear ATP fixation sites, the hydrolysis of which could provide the energy required for a transmembrane transport. In these same regions, some fixation sites for glycosyl groups could be identified. Using a protein sequence data bank, a comparison of the amino acid sequence of these P-glycoproteins and of many other known proteins revealed a strong homology to bacterial membrane transport proteins. Thus, it was concluded that the P-glycoprotein is most probably a membrane protein involved in a transmembrane transport by ferruing a pore or channel through the cellular membrane, s," Kartner and Ling proposed that the natural physiological role played by the P-glycoprotein could consist in a protection mechanism of the cell against toxins by excreting them through the membrane after their penetration inside the cell] Such a detoxification mechanism was already observed in the case of some microorganisms surviving in a medium in competition with other microorganisms producing antibiotics. Other natural roles, as suggested by the same authors, could be the transport of nutrients through the cellular membrane and/or the secretion of proteic or steroid hormones. Interestingly, the P-glycoprotein is normally expressed in kidney cells, liver cells, in cells of the adrenal glands, in parts of the gastrointestinal tract and in the endothelium of the brain of the normal adult, as well as in the placenta. Soon after these first discoveries, it could be demonstrated that the acquired multidrug resistance (mdr) phenotype of cultivated mouse fibroblasts of the NIH 3T3 cell line was linked to the transformation of these cells with DNA segments of high molecular weight harbouring many copies of the human P-glycoprotein gene extracted from resistant human KB carcinoma cells9 This gene was therefore called mdrl (6 and references therein). The mdrl gene copies were expressed at a high level and a correlation between the level of expression, the gene copy number (the degree of amplification) and the efficiency of drug resistance could be established. In another study, it could be shown that the insertion of a full-length cDNA for the human 9
7
9
mdrl gene in a retroviral expression vector infecting non-resistant mouse NIH 3T3 and human KB cells conferred the complete multidrug-resistance phenotype to these cells. 8 Some studies focused on the mechanisms of other detoxification pathways involved in multidrug resistance and on the effect of the acquirement of multidrug resistance on the expression of other genes. Beside the increased production of P-glycoprotein, an overexpression of an anionic isozy,me of the glutathione Stransferase (GSTzc), a detoxification enzyme, was shown to be one of the other causes of multidrug resistance in human breast cancer MCF7 cells.9 This overexpression however occurs without any amplification of the corresponding gene. In this experiment, a c-DNA probe of the same gene was used in a Northern blot assay to show an increased level of specific m-RNA production. Using the same probe in in situ hybridization, Moscow and his coworkers could map the corresponding gene on chromosome llq13. In resistant MCF7 cells, resistant in vivo as in vitro, they could establish an inverse correlation between the expression of this gene and those of the estrogen receptors. The cells expressing estrogen receptors are thus less protected against antineoplastic agents and vice versa. MCF7 cells exhibiting a mdr phenotype loose their mitotic response and sensitivity towards exogen estrogen hormones, which normally produce an elevated amount of the progesterone receptor and increase the secretion of specific proteins.~~ They develop a cross-resistance against the anti-estrogen drug 4-hydroxy tamoxifen and show a hormone-independent growth in vivo in nude mice. While reducing their expression of the estradiol receptor, they acquire an increased level of expression of the epidermic growth factor receptor. By cotransfecting, in one single expression vector system, the human mdrl gene and the gene of the precursors of the human or murine cathepsin L into cultiv a t e d NIH 3T3 m o u s e f i b r o b l a s t cells, the coamplification of the latter genes could be induced, ix Cathepsin L is an acidic protease secreted by cells exhibiting the neoplasic phenotype and responsible for tumorigenesis. Treating these transfected cells with colchicin and selecting resistant cells by progressively inc r e a s e d d o s e s of this toxin r e s u l t s in the co-amplification and overexpression of the gene of the MEP (major excreted protein), the 39 KD precursor of cathepsin L, as well as in its secretion in large amounts, as observed in the case of the acquisition of the normal transformation phenotype in cancer. As investigations of the mdr genes went on, it was found that at least some of them are expressed in a tissue-specific manner in normal tissues. 12Using a DNA segment harbouring the human mdrl gene as a specific probe to analyse m-RNAs e~racted from different cell lines, it could be shown that there is a high expression
MultidnLg R e s i s t a n c e 23
of the human mdrl gene in the adrenal glands, the kidneys, and some cancers after chemotherapy, but a medium expression in the lung, the liver, the colon, the rectum, and, finally, a weak expression in many other tissues. 13 This finding emphasizes that there must be a well organized regulation of the expression of these genes. More recently, a regulating element, possibly a transcriptional enhancer, located some 10 Kilobase upstream of the human mdrl gene, could be identified and shown to markedly augment transcription of this gene regardless of its orientation in a tissue-specific manner, at least in some adrenal and kidney derived cell lines. 14 In an A + T-rich intron of the central region of the same gene, we recently identified an element with a 32bp stretch of alternating purine and pyrimidine bases, which could possibly adopt a left-handed Z D N A conformation and might contribute to the regulation of the . . . . . . . 15 mltlatlon or elongation of transcription. The development of resistance could be linked to an alteration of the control of the mdr gene expression. Interestingly, it was found that an increase of the expression of the human mdrl gene and establishment of a primary resistance to several drugs already occurs before the onset of a gene amplification event. Shcn and h~s coworkers suggested that an activation of the mdrl gene by mutations or epigenetic changes might lead to this phenomenon. T h e r e was a l r e a d y a r e p o r t of a restriction fragment length polymorphism ( R F L P ) associated with 17 the human mdrl gene. The same observation was made in the case of a n o t h e r human multidrug resistance phenotype. The gene involved was called mdr2 and is a member of the human mdr gene family located, as mdrl, on c h r o m o s o m e 7. ~s Over the next few years, even more detailed insights into the genetics and molecular biology of multidrug resistance will allow a better understanding of its basic functions. In the field of oncology new strategies to circumvent this side-effect of anticancer chemotherapy which clinically often leads to failure of the latter are already underway. A first novel therapeutic strategy could be developed using a monoclonal antibody raised against the P-glycoprotein and conjugated to the Pseud o m o n a s toxin. It could be successfully used to specifically kill h u m a n KB carcinoma cells exhibiting an acquired mdr phenotype after chemotherapy with antineoplasic drugs. ~9 In patients, the treatment of advanced and refractory cancer could be achieved using some anticancer drugs in combination with verapamil which acts at least in part by inhibitive competition for the efflux-pump P-glycoprotein. 2~zz We can hope for the development of other strategies working at the level of the molecular control of the expression of multidrug resistance genes in the coming years. Grant support by CRP-Sant6, Luxcmbourg and Societd RCMS, Luxembourg.
Acknowledgement-
REFERENCES 1.
Borst P: DNA amplification and multidrug resistance. Nature 309, 580 (1984).
2.
3. 4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
Riordan J R, Deuchars K, Kartner N, Alon N, Trent J, Ling V: Amplification of P-glycoprotein genes in multidrug-resistant mammalian cell lines. Nature 316, 817 (1985). Kartner N, Ling V: Multidrug resistance in cancer. Sc Am 260, 26 (1989). Seotto K W, Biedler J L, Melera P W: Amplification and expression of genes associated with multidrug resistance in mammalian ceils. Science 232, 751 (1986). Gros P, Croop J, Housman D: Mammalian multidrug resistance gene: co replete c D N A sequence indicates s tro ng homology to bacterial transport proteins. Cell 47, 371 (1986). Chen C J, Chin J E, Ueda K, Clark D P, Pastan I, Gottesman M M, Roninson I B: Internal duplication and homology with bacterial transport proteins in the mdrl (P-glycoprotein) gene from multidrug-resistant human cells. Cell 47, 381 (1986). Shen D W, Fojo A, Roninson l B, Chin J E, Soffit R, Pastan I, Gottesman M M: Multidrug resistance of DNAmediated transformants is linked to transfer of the human mdrl gene. Mol Cell Biol 6, 4039 (1986b). Ueda K, Cardarclli C, Gottesman M M, Pastan 1: Expression ofa full-lenth cDNA for the human "MDRI" gene confers resistance to colchicine, doxorubicin, and vinblastine. Proc Nail Acad Sci USA 84, 3004 (1987). Moscow J A, Townsend A J, Goldsmith M E, WhangPeng J, Vickers P J, Poisson R, Legault-Poisson S, Myers C E, Cowan K H: Isolation of the human anionic glutathione S-transferase cDNA and the relation of its gene expression to estrogen-receptor content in primary breast cancer. Proc Nail Acad Sc'i USA 85, 6518 (1988). Vickers P J, Dickson R B, Shoemaker R, Cowan K H: A multidrug-resistant MCF-7 human breast cancer cell line which exhibits cross-resistance to antiestrogens and hormone-independent tumor growth in vivo. Mol Endocrinol 2,886 (1988). Kane S E, Troen B R, Gal S, Ueda K, Pastan l, Gottesman M M: Use of a cloned multidrug resistance gene for coamplification and overproduction of major excreted protein, a transformation-regulated secreted acid protease. Mol Cell Biol 8, 3316 (1988). Croop J M, Raymond M, Haber D, Devault A, Arceci R J, Gros P, Housman D: The three mouse multidrug resistance genes are expressed in a tissue-specific manner in normal mouse tissues. Mol Cell Biol 97 1346 (1989). Fojo A T, Ueda K, Slamon D J, Poplack D G, Gottesman M M, Pastan I: Expression ofa multidrug-resistance gene in human tumors and tissues. Proc Nail Acad Sci USA 84, 265 (1987). Kohno K, Sato S I, Uchiumi T, Takano H, Kato S, Kuwano M: Tissue-specific enhancer of the human multidrug-resistance (MDRI) gene. J Biol Chem 265, 19690 (1990). Pauly M, Kayser 1, Hammarstr6m-Schmitz M, Dicato M, Hentges F and Michels J M: An A+T-rich intron in the central region of the human mdrl gene harbours a 32-bp minisatellite-like sequence with a stretch of alternating
24 Mo Pauly et al.
16.
17.
18.
19.
purines and pyrimidines. Arch Intern Physiol Biochim 99~ B72 (1991). Shen D W, Fojo A, Chin J E, Roninson I B, Richert N, Pastan l, Gottesman M M: Human multidrug-resistant cell lines: increased mdrl expression can precede gene amplification. Science 232, 643 (1986a). Haber D A, Housman D E: Mspl/Hpall polymorphism in the human multidrug resistance gene 1. Nucl Ac Res 17~ 10142 (1989). Choi K, Hake L E, Bowcock A M, Roninson I B, Cavalli-Sforza L L: RFLPs associated with MDR2, a member of the human multidrug resistance gene family mapped to chromosome 7. N u c l A c Res 15, 6305 (1987). Fitzgerald D J, Willingham M C, Cardarelti C O, Hamada H, Tsuruo T, Gottesman M M, Pastan I: A monoclonal antibody-Pseudomonas toxin conjugate that specifically
kills multidrug-resistantcells. Proc Natl Acad Sci USA 84, 4288 (1987). 20. Ozols R F, Cunnion R E, Klecker R Wet al. : Verapamil and adriamycin in the treatment of drug-resistant ovarian cancer patients. J Clin Oncol 5, 641 (1987). 21. Dalton W S, Grogan T M, Meltzer P S: Drug-resistance in multiple myetoma and non-hodgkin's lymphoma: detection of P-glycoprotein and potential circumvention by addition of verapamil to chemotherapy. J Clin Oncol 7, 415 (1989). 22. Ries F, Dicato M: Treatment of advanced and refractory breast cancer with doxorubicin, vincristine and continuous infusion of verapamil. A phase l-II clinical trial. Med Oncol and Tumor Pharmacother 8, 39 (1991).
GENETIC
ASPECTS OF MULTIDRUG
0736-0118/90 $3.00 + .00
RESISTANCE
M A R C P A U L Y , *t F E R N A N D RIES 2 and M A R I O D I C A T O 2 ILaboratoire de recherche sur le cancer et les maladies du sang, Z.1. Grasbusch, L-3370 Leudelange, Grand-Duchy of Luxembourg 2Centre hospitalier de Luxembourg, Ddpartement d'Hemato-Oncologie, Rue Barbld 4, L-1210 Luxembourg (First submitted 1 November 1991." accepted 27 November 1991)
Gene amplification is responsible both for dihydrofolate reductase induced methotrexate resistance, and for the P-glyeoprotein encoding multigene family induced multidrug resistance. The 6 pairs of hydrophobic regions of the P-glycoprotein fold up in a snake-like structure through the lipidic layers of the cell membrane. Other detoxification mechanisms include the glutathione S-transferase 'pi', but without gene amplification. K ~ Words." Muitidrug resistance
Seven years ago, it was shown that the development of cellular resistance to one or several drugs involves the amplification of at least one specific gene) In cultivated cells treated by a low dose of an antimitotic substance, the amplification is achieved by the transiently blocked replication machinery of the cell, producing extra copies of many genes in such circumstances. The maintenance of the drug in the culture media creates a selection pressure which sorts out cells with several copies of one or a few genes allowing to develop resistance against the drug and to survive. For instance, a treatment of cultivated mouse cells with a low dosis of methotrexate (MTX), an inhibitor of the enzyme dihydrofolate reductase (DHFR), can induce a specific amplification of the corresponding DHFR gene (1 and references therein). The copy number of this gene increases with the MTX dosis, as an effect of selection by MTX. Resistance against MTX is then caused by an overproduction of the D H F R enzyme.
cells selected by colchicin and overexpressing this P-glycoprotein. The isolation of positive clones was achieved using a specific monoclonal antibody raised against the P-glycoprotein, in an immunobtot assay. In a further step, a specific cDNA probe of the P-glycoprotein gene was used in Northern blot assays to demonstrate the presence, in resistant cells, of a proportionally increased amount of specific mRNA of about 4.7 Kb. In Southern blot assays performed by Riordan and his coworkers on genomic DNA, the identification of eight specific bands showed the existence of several homologous genes already in nonresistant cells. These bands become even more pronounced in the case of resistant cells. They therefore suggested that these genes probably belong to a P-glycoprotein encoding multigene family, members of which are amplified during the appearance of the multidrug resistance phenotype. In resistant cells, up to 60 copies of each gene could be present.
Multidrug resistant mammalian cells overproduce a glycoprotein of a molecular weight of 170 Kd, after amplification of the corresponding geneY After synthesis, the P-glycoprotein is embedded in the plasma membrane of the cell.
Independently of each other, different laboratories showed that a homologous multigene family also exists in different normal or tumoral cells of other mammals (mouse, rat, man). 3'4 The genes of these families are similarly organized and this fact suggests that they have been well conserved among the different mammalian species, during evolution. Therefore, Kartner and Ling proposed that this P-gtycoprotein gene supra-family was probably generated in two steps: an internal duplication event of a single ancestral gene leads to the formation of a first ancient multigene family. Next, divergence from this basic family occurred at the dawn
The P-glycoprotein encoding part of a cellular gene was isolated by Riordan and his coworkers from a chinese hamster cDNA library, a3 This library was established in the bacteriophage expression vector .;tgtll using mRNAs from resistant chinese hamster ovary 'To whom correspondanceshould be addressed. 21
22 M. Pauly et al. of mammalian appearance. This divergence would then explain the slight structural differences observed among the P-glycoproteins produced in the cells of the different mammalian species. After isolation and cloning of the corresponding gene, the murine and human P-glycoprotein could be obtained in high amounts and sequenced. 5'6 The primary structure of these proteins shows a sequence of about 1,280 amino acids. A hydropathy analysis of the different regions of the proteins allowed to conclude that they are duplicated and contain six pairs of hydrophobic regions. This observation lead to the suggestion that these regions fold up in a snake-like structure and pass through the double lipidic layers of the cellular membrane. Some hydrophilic regions would be located towards the inner and outer cellular compartments and bear ATP fixation sites, the hydrolysis of which could provide the energy required for a transmembrane transport. In these same regions, some fixation sites for glycosyl groups could be identified. Using a protein sequence data bank, a comparison of the amino acid sequence of these P-glycoproteins and of many other known proteins revealed a strong homology to bacterial membrane transport proteins. Thus, it was concluded that the P-glycoprotein is most probably a membrane protein involved in a transmembrane transport by ferruing a pore or channel through the cellular membrane, s," Kartner and Ling proposed that the natural physiological role played by the P-glycoprotein could consist in a protection mechanism of the cell against toxins by excreting them through the membrane after their penetration inside the cell] Such a detoxification mechanism was already observed in the case of some microorganisms surviving in a medium in competition with other microorganisms producing antibiotics. Other natural roles, as suggested by the same authors, could be the transport of nutrients through the cellular membrane and/or the secretion of proteic or steroid hormones. Interestingly, the P-glycoprotein is normally expressed in kidney cells, liver cells, in cells of the adrenal glands, in parts of the gastrointestinal tract and in the endothelium of the brain of the normal adult, as well as in the placenta. Soon after these first discoveries, it could be demonstrated that the acquired multidrug resistance (mdr) phenotype of cultivated mouse fibroblasts of the NIH 3T3 cell line was linked to the transformation of these cells with DNA segments of high molecular weight harbouring many copies of the human P-glycoprotein gene extracted from resistant human KB carcinoma cells9 This gene was therefore called mdrl (6 and references therein). The mdrl gene copies were expressed at a high level and a correlation between the level of expression, the gene copy number (the degree of amplification) and the efficiency of drug resistance could be established. In another study, it could be shown that the insertion of a full-length cDNA for the human 9
7
9
mdrl gene in a retroviral expression vector infecting non-resistant mouse NIH 3T3 and human KB cells conferred the complete multidrug-resistance phenotype to these cells. 8 Some studies focused on the mechanisms of other detoxification pathways involved in multidrug resistance and on the effect of the acquirement of multidrug resistance on the expression of other genes. Beside the increased production of P-glycoprotein, an overexpression of an anionic isozy,me of the glutathione Stransferase (GSTzc), a detoxification enzyme, was shown to be one of the other causes of multidrug resistance in human breast cancer MCF7 cells.9 This overexpression however occurs without any amplification of the corresponding gene. In this experiment, a c-DNA probe of the same gene was used in a Northern blot assay to show an increased level of specific m-RNA production. Using the same probe in in situ hybridization, Moscow and his coworkers could map the corresponding gene on chromosome llq13. In resistant MCF7 cells, resistant in vivo as in vitro, they could establish an inverse correlation between the expression of this gene and those of the estrogen receptors. The cells expressing estrogen receptors are thus less protected against antineoplastic agents and vice versa. MCF7 cells exhibiting a mdr phenotype loose their mitotic response and sensitivity towards exogen estrogen hormones, which normally produce an elevated amount of the progesterone receptor and increase the secretion of specific proteins.~~ They develop a cross-resistance against the anti-estrogen drug 4-hydroxy tamoxifen and show a hormone-independent growth in vivo in nude mice. While reducing their expression of the estradiol receptor, they acquire an increased level of expression of the epidermic growth factor receptor. By cotransfecting, in one single expression vector system, the human mdrl gene and the gene of the precursors of the human or murine cathepsin L into cultiv a t e d NIH 3T3 m o u s e f i b r o b l a s t cells, the coamplification of the latter genes could be induced, ix Cathepsin L is an acidic protease secreted by cells exhibiting the neoplasic phenotype and responsible for tumorigenesis. Treating these transfected cells with colchicin and selecting resistant cells by progressively inc r e a s e d d o s e s of this toxin r e s u l t s in the co-amplification and overexpression of the gene of the MEP (major excreted protein), the 39 KD precursor of cathepsin L, as well as in its secretion in large amounts, as observed in the case of the acquisition of the normal transformation phenotype in cancer. As investigations of the mdr genes went on, it was found that at least some of them are expressed in a tissue-specific manner in normal tissues. 12Using a DNA segment harbouring the human mdrl gene as a specific probe to analyse m-RNAs e~racted from different cell lines, it could be shown that there is a high expression
MultidnLg R e s i s t a n c e 23
of the human mdrl gene in the adrenal glands, the kidneys, and some cancers after chemotherapy, but a medium expression in the lung, the liver, the colon, the rectum, and, finally, a weak expression in many other tissues. 13 This finding emphasizes that there must be a well organized regulation of the expression of these genes. More recently, a regulating element, possibly a transcriptional enhancer, located some 10 Kilobase upstream of the human mdrl gene, could be identified and shown to markedly augment transcription of this gene regardless of its orientation in a tissue-specific manner, at least in some adrenal and kidney derived cell lines. 14 In an A + T-rich intron of the central region of the same gene, we recently identified an element with a 32bp stretch of alternating purine and pyrimidine bases, which could possibly adopt a left-handed Z D N A conformation and might contribute to the regulation of the . . . . . . . 15 mltlatlon or elongation of transcription. The development of resistance could be linked to an alteration of the control of the mdr gene expression. Interestingly, it was found that an increase of the expression of the human mdrl gene and establishment of a primary resistance to several drugs already occurs before the onset of a gene amplification event. Shcn and h~s coworkers suggested that an activation of the mdrl gene by mutations or epigenetic changes might lead to this phenomenon. T h e r e was a l r e a d y a r e p o r t of a restriction fragment length polymorphism ( R F L P ) associated with 17 the human mdrl gene. The same observation was made in the case of a n o t h e r human multidrug resistance phenotype. The gene involved was called mdr2 and is a member of the human mdr gene family located, as mdrl, on c h r o m o s o m e 7. ~s Over the next few years, even more detailed insights into the genetics and molecular biology of multidrug resistance will allow a better understanding of its basic functions. In the field of oncology new strategies to circumvent this side-effect of anticancer chemotherapy which clinically often leads to failure of the latter are already underway. A first novel therapeutic strategy could be developed using a monoclonal antibody raised against the P-glycoprotein and conjugated to the Pseud o m o n a s toxin. It could be successfully used to specifically kill h u m a n KB carcinoma cells exhibiting an acquired mdr phenotype after chemotherapy with antineoplasic drugs. ~9 In patients, the treatment of advanced and refractory cancer could be achieved using some anticancer drugs in combination with verapamil which acts at least in part by inhibitive competition for the efflux-pump P-glycoprotein. 2~zz We can hope for the development of other strategies working at the level of the molecular control of the expression of multidrug resistance genes in the coming years. Grant support by CRP-Sant6, Luxcmbourg and Societd RCMS, Luxembourg.
Acknowledgement-
REFERENCES 1.
Borst P: DNA amplification and multidrug resistance. Nature 309, 580 (1984).
2.
3. 4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
Riordan J R, Deuchars K, Kartner N, Alon N, Trent J, Ling V: Amplification of P-glycoprotein genes in multidrug-resistant mammalian cell lines. Nature 316, 817 (1985). Kartner N, Ling V: Multidrug resistance in cancer. Sc Am 260, 26 (1989). Seotto K W, Biedler J L, Melera P W: Amplification and expression of genes associated with multidrug resistance in mammalian ceils. Science 232, 751 (1986). Gros P, Croop J, Housman D: Mammalian multidrug resistance gene: co replete c D N A sequence indicates s tro ng homology to bacterial transport proteins. Cell 47, 371 (1986). Chen C J, Chin J E, Ueda K, Clark D P, Pastan I, Gottesman M M, Roninson I B: Internal duplication and homology with bacterial transport proteins in the mdrl (P-glycoprotein) gene from multidrug-resistant human cells. Cell 47, 381 (1986). Shen D W, Fojo A, Roninson l B, Chin J E, Soffit R, Pastan I, Gottesman M M: Multidrug resistance of DNAmediated transformants is linked to transfer of the human mdrl gene. Mol Cell Biol 6, 4039 (1986b). Ueda K, Cardarclli C, Gottesman M M, Pastan 1: Expression ofa full-lenth cDNA for the human "MDRI" gene confers resistance to colchicine, doxorubicin, and vinblastine. Proc Nail Acad Sci USA 84, 3004 (1987). Moscow J A, Townsend A J, Goldsmith M E, WhangPeng J, Vickers P J, Poisson R, Legault-Poisson S, Myers C E, Cowan K H: Isolation of the human anionic glutathione S-transferase cDNA and the relation of its gene expression to estrogen-receptor content in primary breast cancer. Proc Nail Acad Sc'i USA 85, 6518 (1988). Vickers P J, Dickson R B, Shoemaker R, Cowan K H: A multidrug-resistant MCF-7 human breast cancer cell line which exhibits cross-resistance to antiestrogens and hormone-independent tumor growth in vivo. Mol Endocrinol 2,886 (1988). Kane S E, Troen B R, Gal S, Ueda K, Pastan l, Gottesman M M: Use of a cloned multidrug resistance gene for coamplification and overproduction of major excreted protein, a transformation-regulated secreted acid protease. Mol Cell Biol 8, 3316 (1988). Croop J M, Raymond M, Haber D, Devault A, Arceci R J, Gros P, Housman D: The three mouse multidrug resistance genes are expressed in a tissue-specific manner in normal mouse tissues. Mol Cell Biol 97 1346 (1989). Fojo A T, Ueda K, Slamon D J, Poplack D G, Gottesman M M, Pastan I: Expression ofa multidrug-resistance gene in human tumors and tissues. Proc Nail Acad Sci USA 84, 265 (1987). Kohno K, Sato S I, Uchiumi T, Takano H, Kato S, Kuwano M: Tissue-specific enhancer of the human multidrug-resistance (MDRI) gene. J Biol Chem 265, 19690 (1990). Pauly M, Kayser 1, Hammarstr6m-Schmitz M, Dicato M, Hentges F and Michels J M: An A+T-rich intron in the central region of the human mdrl gene harbours a 32-bp minisatellite-like sequence with a stretch of alternating
24 Mo Pauly et al.
16.
17.
18.
19.
purines and pyrimidines. Arch Intern Physiol Biochim 99~ B72 (1991). Shen D W, Fojo A, Chin J E, Roninson I B, Richert N, Pastan l, Gottesman M M: Human multidrug-resistant cell lines: increased mdrl expression can precede gene amplification. Science 232, 643 (1986a). Haber D A, Housman D E: Mspl/Hpall polymorphism in the human multidrug resistance gene 1. Nucl Ac Res 17~ 10142 (1989). Choi K, Hake L E, Bowcock A M, Roninson I B, Cavalli-Sforza L L: RFLPs associated with MDR2, a member of the human multidrug resistance gene family mapped to chromosome 7. N u c l A c Res 15, 6305 (1987). Fitzgerald D J, Willingham M C, Cardarelti C O, Hamada H, Tsuruo T, Gottesman M M, Pastan I: A monoclonal antibody-Pseudomonas toxin conjugate that specifically
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