Abstract
Background
Cystic fibrosis (CF) is the most common lethal recessive disease affecting children in the U.S. and Europe. For this reason, a number of ongoing attempts are being made to treat the disease either by gene therapy or pharmacotherapy. Several phase 1 gene therapy trials have been completed, and a phase 2 clinical trial with the xanthine drug CPX is in progress. The protein coded by the principal CFTR mutation, ΔF508-CFTR, fails to traffic efficiently from the endoplasmic reticulum to the plasma membrane, and is the pathogenic basis for the missing cAMP-activated plasma membrane chloride channel. CPX acts by binding to the mutant ΔF508-CFTR and correcting the trafficking deficit. CPX also activates mutant CFTR channels. The comparative genomics of wild-type and mutant CFTR has not previously been studied. However, we have hypothesized that the gene expression patterns of human cells expressing mutant or wild-type CFTR might differ, and that a drug such as CPX might convert the mutant gene expression pattern into one more characteristic of wild-type CFTR. To the extent that this is true, a pharmacogenomic profile for such corrective drugs might be deduced that could simplify the process of drug discovery for CF.
Materials and Methods
To test this hypothesis we used cDNA microarrays to study global gene expression in human cells permanently transfected with either wild-type or mutant CFTR. We also tested the effects of CPX on global gene expression when incubated with cells expressing either mutant or wild-type CFTR.
Results
Wild-type and mutant ΔF508-CFTR induce distinct and differential changes in cDNA microarrays, significantly affecting up to 5% of the total genes in the array. CPX also induces substantial mutation-dependent and -independent changes in gene expression. Some of these changes involve movement of gene expression in mutant cells in a direction resembling expression in wild-type cells.
Conclusions
These data clearly demonstrate that cDNA array analysis of cystic fibrosis cells can yield useful pharmacogenomic information with significant relevance to both gene and pharmacological therapy. We suggest that this approach may provide a paradigm for genome-based surrogate endpoint testing of CF therapeutics prior to human administration.
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References
Bailey DS, Bondar A, Furness LM. (1998) Pharmacogenomics—it’s not just pharmacogenetics. Curr. Opin. Biotechnol 6: 595–601.
Ferrari P. (1998) Pharmacogenomics: a new approach to individual therapy of hypertension? Curr. Opin. Nephrol. Hypertens. 7: 217–221.
Graever G, Shoemaker DD, Jones TW, et al. (1999) Genomic profiling of drug sensitivities via induced haploinsufficiency. Nat. Genet. 3: 278–283.
Welsh MJ, Tsui L-C, Boat TF, Beaudet AL. (1995) Cystic fibrosis. In: Scriver CL, Sly WS, Valle D (eds). The Metabolic and Molecular Bases of Inherited Diseases, 7th ed. McGraw-Hill, New York, pp. 3799–3876.
Collins FS. (1992) Cystic fibrosis molecular biology and therapeutic implications. Science 256: 774–779.
Drumm ML, Pope HA, Cliff WH, et al. (1990) Correction of the cystic fibrosis defect in vitro by retrovirus-mediated gene transfer. Cell 62: 1227–1233.
Rubenstein RC, Egan ME, Zeitlin PL. (1997) In vitro pharmacologic restoration of CFTR-mediated chloride transport in sodium 4-phenylbutyrate in cystic fibrosis epithelial cells containing delta F508-CFTR. J. Clin. Invest. 100: 2457–2465.
Pollard HB. (1997) Role of CPX in promoting trafficking and chloride channel activity of wildtype and mutant CFTR. Pediatr. Pulmonol. S14: 128–131.
Riordan JR, Rommens JM, Karem B-S, et al. (1989) Identification of the cystic fibrosis gene: Cloning and characterization of complementary DNA. Science 245: 1066–1073.
Rommens JM, Iannuzzi MC, Karem B-S, et al. (1989) Identification of the cystic fibrosis gene: chromosome walking and jumping. Science 245: 1059–1065.
Karem B-S, Rommens JM, Buchanan JA, et al. (1989) Identification of the cystic fibrosis gene: genetic analysis. Science 245: 1073–1080.
Tsui L-C. (1992) Mutations and sequence variations detected in the cystic fibrosis transmembrane conductance regulator (CFTR) gene: a report from the cystic fibrosis genetic analysis consortium. Hum. Mutat. 1: 197–203.
Tsui L-C. (1992) The spectrum of cystic fibrosis mutations. Trends Genet. 8: 392–398.
Schultz BD, Singh AK, Devor DC, Bridges RJ. (1999) Pharmacology of CFTR chloride channel activity. Physiol. Rev. 79: S109–S144.
Eidelman O, Guay-Broder C, van Galen PJM, et al. (1992) A1-adenosine antagonists activate chloride efflux from cystic fibrosis cells. Proc. Natl. Acad. Sci. (U.S.A.) 89: 5562–5566.
Schweibert E, Gruenert D, Stanton B. (1992) G-proteins inhibit cAMP-activated chloride channels in normal and CF epithelia. Pediatr. Pulmonol. S8: 257.
Guay-Broder C, Jacobson KA, BarNoy S, et al. (1995) A1-receptor antagonist 8 cyclopentyl-1,3-dipropyl-xanthine (CPX) selectively activates chloride efflux from human epithelial and mouse fibroblast cell lines expressing the CFTR(ΔF508) mutation, but not the wild type CFTR. Biochemistry 34: 9079–9087.
Jacobson KA, Guay-Broder C, van Galen PJM et al. (1995) Stimulation of alkylxanthines of chloride efflux from CFPAC-1 cells does not involve A1-adenosine receptors. Biochemistry 34: 9088–9094.
Casavola V, Turner RJ, Guay-Broder C, Jacobson KA, Eidelman O, Pollard HB. (1995) CPX, a selective A1 adenosine receptor antagonist, regulates intracellular pH in cystic fibrosis cells. Am. J. Physiol. (Cell) 269: C226–233.
Haws CM, Nepomuceno I, Krouse ME, et al. (1996) ΔF508-CFTR channels: kinetics, activation by forskolin, and potentiation by xanthines. Am. J. Physiol. 270: 1544–1555.
Arispe N, Ma J, Jacobson KA, Pollard HB. (1998) Direct activation of cystic fibrosis transmembrane conductance regulator by 8-cyclopentyl-l,3-dipropylxanthine and 1,3-diallyl-8-cyclohexyl-xanthine. J. Biol. Chem. 273: 5724–5734.
Cheng SH, Gregory RJ, Marshall J, et al. (1990) Defective intracellular transport and processing of CFTR is the molecular basis of most cystic fibrosis. Cell 63: 827–834.
Cohen BE, Lee G, Jacobson KA, et al. (1997) CPX (1,3-dipropyl-8-cyclopentyl xanthine) and other alkyl-xanthines differentially bind to the wild type and ΔF508 mutant first nucleotide binding fold (NBF-1) domains of the cystic fibrosis transmembrane conductance regulator (CFTR). Biochemistry 36: 6455–6461.
Stutts MJ, Canessa CM, Olsen JC, et al. (1995) CFTR as a cAMP-dependent regulator of sodium channels. Science 269: 847–850.
Ismailov II, Awayda MS, Jovov B, et al. (1996) Regulation of epithelial sodium channels by the cystic fibrosis transmembrane conductance regulator. J. Biol Chem. 271: 4725–4732.
Schweibert EM, Benos DJ, Egan ME, Stutts MJ, Guggino WB. (1999) CFTR is a conductance regulator as well as a chloride channel. Physiol. Rev. 79: S145–S166.
Jacobson KA. (1988) Adenosine A3 receptors: novel ligands and paradoxical effects. Trends Pharmacol. Sci. 19: 184–191.
Fulmer SB, Schweibert EM, Morales MM, Guggino WB, Cutting GR. (1995) Two cystic fibrosis transmembrane conductance regulator mutations have different effects on both pulmonary phenotype and regulation of outwardly rectified chloride currents. Proc. Natl. Acad. Sci. U.S.A. 92: 6832–6836.
Neglia JP, FitzSimmons SC, Maisonneuve P, et al. (1995) The risk of cancer among patients with cystic fibrosis. Cystic Fibrosis and Cancer Study Group. N. Engl. J. Med. 332: 494–499.
Sheldon CD, Hodson ME, Carpenter LM, Swerdlow AJ. (1993) A cohort study of cystic fibrosis and malignancy. Br. J. Cancer 68: 1025–1028.
Chaun H, Paty B, Nakiela EM, Schmidt N, Holden JK, Melosky B. (1996) Colonic carcinoma in two adult cystic fibrosis patients. Can. J. Gastroenterol. 10: 440–442.
Neugut AI, Jacobson JS, Suh S, Mukherjee R, Arber N. (1998) The epidemiology of cancer of the small bowel. Cancer Epidemiol. Biomark. Prev. 7: 243–251.
Tsogalis GJ, Faber G, Dalldorf FG, Friedman KJ, Silverman LM, Yankaskas JR. (1994) Association of pancreatic adenocarcinoma, mild lung disease, and delta F508 mutation in a cystic fibrosis patient. Clin. Chem. 40: 1972–1974.
Garcia FU, Galindo LM, Holsclaw DS, Jr. (1998) Breast abnormalities in patients with cystic fibrosis: previously unrecognised changes. Ann. Diagn. Pathol. 2: 281–285.
Southey MC, Batten L, Anderson CR, et al. (1998) CFTR deltaF508 carrier status, risk of breast cancer before the age of 40 and histological grading in a population-based case-control study. Int. J. Cancer 79: 487–489.
Padua RA, Warren N, Grimshaw D, et al. (1997) The cystic fibrosis delta F508 mutation and cancer. Hum. Mutat. 10: 45–48.
Gress TM, Muller-Pillasch F, Geng M, et al. (1996) A pancreatic cancer-specific expression profile. Oncogene 13: 1819–1830.
Heller RA, Schena M, Chai A, et al. (1997) Discovery and analysis of inflammatory disease-related genes using cDNA microarrays. Proc. Natl. Acad. Sci. U.S.A. 94: 2150–2155.
Pappalardo PA, Bonner R, Krizman DB, Emmert-Buck MR, Liotta LA. (1998) Microdissection, microchip arrays, and molecular analysis of tumor cells (primary and metastases). Semin. Radiat. Oncol. 8: 217–223.
Pietu G, Alibert O, Guichard V, et al. (1996) Novel gene transcripts preferentially expressed in human muscles revealed by quantitative hybridization of a high density cDNA array. Genome Res. 6: 492–503.
Schena M, Shalon D, Heller R, Chai A, Brown PO, Davis RW. (1996) Parallel human genome analysis: microarray-based expression monitoring of 1000 genes. Proc. Natl. Acad. Sci. U.S.A. 93: 10614–10619.
Welford SM, Gregg J, Chen E, et al. (1998) Detection of differentially expressed genes in primary tumor tissues using representational differences analysis coupled to microarray hybridization. Nucl. Acids Res. 26: 3059–3065.
Lipshutz RJ, Fodor SP, Gingeras TR, Lockhart DJ. (1999) High density synthetic oligonucleotide arrays. Nat. Genet. 21 (1 Suppl): 20–24.
Watson SJ, Akil H. (1999) Gene chips and arrays revealed: a primer on the power and their uses. Biol. Psychiatry 45: 533–543.
Alizadeh A, Eisen M, Botstein D, Brown PO, Staudt LM. (1998) Probing lymphocyte biology by genome-scale gene expression analysis. J. Clin. Immunol. 18: 373–379.
DeRisi J, Penland L, Brown PO, et al. (1996) Use of a cDNA microarray to analyse gene expression patterns in human cancer. Nat. Genet. 14: 367–370.
Ollila J, Vihinen M. (1998) Stimulation of B and T cells activates expression of transcription and differentiation factors. Biochem. Biophys. Res. Commun. 249: 475–480.
Shim C, Zhang W, Rhee CH, Lee JH. (1998) Profiling of differentially expressed genes in human primary cervical cancer by complementary DNA expression array. Clin. Cancer Res. 4: 3045–3050.
Tao T, Xie J, Drumm ML, Zhao J, Davis PB, Ma J. (1996) Slow conversions among subconductance states of cystic fibrosis transmembrane conductance regulator chloride channel. Biophys. J. 70: 743–753.
Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ. (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18: 5294–5299.
Gadsby DC, Nairn AC. (1999) Control of CFTR channel gating by phosphorylation and nucleotide hydrolysis. Physiol. Rev. 79: S77–S107.
Gao Z, Chen T, Weber MJ, Linden J. (1999) AaBBadenosine and P2Y2 receptors stimulate mitogen-activated protein kinase in human embryonic kidney-293 cells. J. Med. Chem. 274: 5972–5980.
Shimegi S. (1998) Mitogenic action of adenosine on osteoblast-like cells, MC3T3-E1. Calcif. Tissue Int. 62: 418–425.
Yuh IS, Sheffield LG. (1998) Adenosine stimulation of DNA synthesis in mammary epithelial cells. Proc. Soc. Exp. Biol. Med. 218: 341–348.
Lelievre V, Muller JM, Falcon J. (1998) Adenosine modulates cell proliferation in human colonic adenocarcinoma. I. Possible involvement of A1 receptor subtypes in HT29 cells. Eur. J. Pharmacol. 341: 289–297.
Bassen DE Jr, Eisen MB, Boguski MS. (1999) Gene expression informatics—it’s all in your mine. Nat. Genet. Suppl. 21: 51–55.
Acknowledgments
The authors thank Dr. A. Namboodiri, Dr. S. Galdwicky, Dr. L. Fossom, Dr. X. Leighton, and Ms. M. Glasman for substantive help and discussions, and acknowledge financial support from the NIH (RO1-DK53051), the Cystic Fibrosis Foundation, and the Juvenile Diabetes Foundation, International.
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Srivastava, M., Eidelman, O. & Pollard, H.B. Pharmacogenomics of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and the Cystic Fibrosis Drug CPX Using Genome Microarray Analysis. Mol Med 5, 753–767 (1999). https://doi.org/10.1007/BF03402099
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DOI: https://doi.org/10.1007/BF03402099