Method of obtaining purified enterochromaffin cells and methods of using same
(19) AUSTRALIAN PATENT OFFICE (54) (51)6 (21) (87) (30) (31) (43) (43) (71) (72) Title Method of obtaining purified enterochromaffin cells and methods of using same International Patent Classification(s) C12Q 001/68 C12N 005/06 G01N 033/567 Application No: 2002351413 WIPO No: WO03/060147 Priority Data (22) Application Date: 2002.12.23 Number 60/342,958 (32) Date (33) Country US 200510277 2001 .12.21 Publication Date : 2003 .07.30 Publication Journal Date : 2003 .09.04 Applicant(s) MODLIN, Imn; SACHS, George; BRUGGEMAN, Patrick Inventor(s) Sachs, Georiie; Modlin, Irvin; Bruggeman, Patrick -1- Substantially purified enterochromaffin (EC) cells and methods of using the cells to identify nucleic acid sequences involved in disorders associated with enterochromaffin cells are provided. In addition, methods of treating enterochromaffin cell-associated disorders such as carcinoid tumor, carcinoid syndrome and irritable bowel disorder by targeting the identified nucleic acid sequences are provided. What is claimed is: 1. A method of preparing substantially purified enterochromaffin (EC) cells, comprising: a) obtaining a mixed population of cells containing EC cells from a subject; b) separating the population of cells by a separation technique based on density of tl cells, size of the cells, or both, wherein a separated population is formed, the separated population comprising EC cells that are separated from other cells often- population, thereby preparing substantially purified EC cells.
2. The method of claim 1, further comprising: c) removing the EC cells from the separated population, thereby obtaining substantially purified EC cells.
3. The method of claim 1, wherein the separation technique comprises density gradient centrifugation.
4. The method of claim 3, wherein the density gradient centrifugation comprises a continuous gradient.
5. The method of claim 3, wherein the density gradient centrifugation comprises a discontinuous gradient.
6. The method of claim 1, wherein the separation technique comprises elutriation.
7. The method of claim 1 wherein the separation technique comprises elutriation, followed by density gradient centrifugation.
8. The method of claim 1, wherein the separating step comprises fluorescence activated cell sorting. <Desc/Clms Page number 37> 9. A method of obtaining an isolated enterochromaffin (EC) cell, comprising: a) contacting a population of cells containing EC cells with a reagent that specificall binds EC cells; and b) isolating cells specifically bound by the reagent, thereby obtaining an isolated EC cell.
10. The method of claim 9, the method further comprising, before the isolating step, detectin cells specifically bound by the reagent.
11. The method of claim 10, wherein the detecting is performed using an immunocytochemical method, a histochemical method or in situ hybridization.
12. The method of claim 9, wherein the reagent comprises an antibody that specifically binds a hormone synthesized or secreted by the EC cell.
13. The method of claim 12, wherein the hormone is serotonin, histamine, chromagranin A, VMAT 1, VMAT 2 or guanylin.
14. The method of claim 9, wherein the reagent comprises an antibody that specifically binds a synthetic enzyme used in the biosynthesis of hormones by EC cells.
15. The method of claim 14, wherein the synthetic enzyme is tryptophan hydroxylase, histidine decarboxylase or other synthetic enzyme used in the biosynthesis of hormones by EC cells.
16. The method of claim 9, wherein the reagent comprises an antibody that specifically binds an EC cell surface receptor, and detecting the antibody specifically bound to the EC cell.
17. The method of claim 16, wherein the EC cell receptor comprises a CCK-A or CCK-B receptor.
18. The method of claim 9, wherein the reagent comprises an oligonucleotide that specificall) binds a nucleic acid sequence encoding tryptophan hydroxylase or guanylin.
19. The method of claim 9, wherein the isolating is by laser capture microscopy. <Desc/Clms Page number 38> 20. A method of identifying a polynucleotide associated with an enterochromaffin (EC) cell- associated disorder, comprising: a) contacting an EC cell from a subject having or suspected of having an EC cell- associated disorder with an array of probes representative of EC cell nucleic acid molecules expressed in EC cells from a subject having the EC cell-associated disorder; and b) detecting expression of nucleic acid molecules in an EC cell of the subject, wherein expression of nucleic acid molecules in the EC cell of the subject differs from th expression of nucleic acid molecules in an EC cell of a subject not having the disorder, thereby identifying a polynucleotide associated with an EC cell-associated disorder in an EC cell.
21. The method of claim 20, wherein the EC cell-associated disorder comprises abnormal neuroendocrine hormone expression of an EC cell.
22. The method of claim 21, wherein the neuroendocrine hormone expressed comprises serotonin, histamine, VIP, glucagons, somatostatin, neurotensin, guanylin or a combination thereof.
23. The method of claim 20, wherein the EC cell-associated disorder comprises a carcinoid tumor, carcinoid syndrome or irritable bowel syndrome.
24. The method of claim 20, wherein the wherein the EC cell-associated disorder comprises an EC cell proliferative disorder.
25. The method of claim 20, wherein the level of expression in an EC cell from a subject not having an EC cell-associated disorder is lower than the level of expression in EC cells from a subject having an EC cell-associated disorder.
26. The method of claim 20, wherein the EC cell comprises a gastric EC cell, intestinal EC cell, colorectal EC cell, or appendical EC cell. <Desc/Clms Page number 39> 27. A method of identifying a polynucleotide associated with enterochromaffin (EC) cell proliferation, comprising: a) contacting a test EC cell with an array of probes representative of EC cell nucleic acid molecules expressed in normal EC cells; b) detecting expression of nucleic acid molecules in the test EC cell; c) performing a clustering analysis of the nucleic acid molecules expressed in the tes EC cell; and d) detecting a cluster comprising polynucleotides associated with proliferation of EC cells ; thereby identifying a polynucleotide associated with EC cell proliferation.
28. The method of claim 27, wherein the clustering is hierarchical clustering.
29. The method of claim 27, further comprising isolating the polynucleotide associated with EC cell proliferation.
30. An isolated polynucleotide associated with EC cell proliferation, the polynucleotide isolated by the method of claim 29.
31. A method of treating or preventing an EC cell-associated disorder, comprising administering to a subject having or suspected of having an EC cell-associated disorder an agent that modulates expression of a polynucleotide associated with an EC cell-associated disorder.
32. The method of claim 31, wherein the EC cell-associated disorder comprises abnormal neuroendocrine hormone expression of an EC cell.
33. The method of claim 31, wherein the wherein the EC cell-associated disorder comprises an EC cell proliferative disorder.
34. The method of claim 31, wherein the agent decreases expression of the polynucleotide.
35. The method of claim 31, wherein the agent increases expression of the polynucleotide.
36. A composition, comprising an antisense polynucleotide that specifically binds a mRNA encoding a polynucleotide associated with an EC cell-associated disorder, and a carrier. <Desc/Clms Page number 40> 37. The composition of claim 36, wherein the composition is a pharmaceutical composition.
38. A cDNA library prepared from the EC cells of claim 1.
39. The cDNA library of claim 38, wherein the EC cells are obtained from a subject having a EC cell-associated disorder and exhibit altered phenotypic expression.
40. A method of treating or preventing fibrosis, comprising administering to a subject having or suspected of having fibrosis an agent that modulates expression of a factor essential for the production of fibrosis.