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• W2003706637 abstract "The nucleoporin Nup98 gene is frequently rearranged in acute myelogenous leukemia (AML). In most cases this results in fusion of the N terminus of Nup98 to the DNA binding domain of a homeodomain transcription factor. The prototype of these fusions, Nup98-HOXA9, is associated with human AML and induces AML in mouse models. To understand the mechanisms by which Nup98-HOXA9 causes AML, we expressed it in myeloid cells and identified its target genes using high density oligonucleotide microarrays. The analysis was performed in triplicate and was confirmed by quantitative real time PCR. Of the 102 Nup98-HOXA9 target genes identified, 92 were up-regulated, and only 10 were down-regulated, suggesting a transcriptional activation function. A similar analysis of wild-type HOXA9 revealed 13 target genes, 12 of which were up-regulated, and 1 was down-regulated. In contrast, wild-type Nup98 had no effect on gene expression, demonstrating that the HOXA9 DNA binding domain is required for gene regulation. Co-transfection experiments using a luciferase reporter linked to the promoter of one of the Nup98-HOXA9 target genes confirmed up-regulation at the transcriptional level by Nup98-HOXA9 but not by either HOXA9 or Nup98. These data indicate that Nup98-HOXA9 is an aberrant transcription factor whose activity depends on the HOXA9 DNA binding domain but has a stronger and wider transcriptional effect than HOXA9. Several of the genes regulated by Nup98-HOXA9 are associated with increased cell proliferation and survival as well as drug metabolism, providing insights into the pathogenesis and epidemiology of Nup98-HOXA9-induced AML. The nucleoporin Nup98 gene is frequently rearranged in acute myelogenous leukemia (AML). In most cases this results in fusion of the N terminus of Nup98 to the DNA binding domain of a homeodomain transcription factor. The prototype of these fusions, Nup98-HOXA9, is associated with human AML and induces AML in mouse models. To understand the mechanisms by which Nup98-HOXA9 causes AML, we expressed it in myeloid cells and identified its target genes using high density oligonucleotide microarrays. The analysis was performed in triplicate and was confirmed by quantitative real time PCR. Of the 102 Nup98-HOXA9 target genes identified, 92 were up-regulated, and only 10 were down-regulated, suggesting a transcriptional activation function. A similar analysis of wild-type HOXA9 revealed 13 target genes, 12 of which were up-regulated, and 1 was down-regulated. In contrast, wild-type Nup98 had no effect on gene expression, demonstrating that the HOXA9 DNA binding domain is required for gene regulation. Co-transfection experiments using a luciferase reporter linked to the promoter of one of the Nup98-HOXA9 target genes confirmed up-regulation at the transcriptional level by Nup98-HOXA9 but not by either HOXA9 or Nup98. These data indicate that Nup98-HOXA9 is an aberrant transcription factor whose activity depends on the HOXA9 DNA binding domain but has a stronger and wider transcriptional effect than HOXA9. Several of the genes regulated by Nup98-HOXA9 are associated with increased cell proliferation and survival as well as drug metabolism, providing insights into the pathogenesis and epidemiology of Nup98-HOXA9-induced AML. Most cases of AML 1The abbreviations used are: AML, acute myelogenous leukemia; FG, phenylalanine-glycine; MSCV, murine stem cell virus; IRES, internal ribosomal entry site; GFP, green fluorescent protein; MALT, mucosa-associated lymphoid tissue; MEF, myocyte enhancer factor; FBS, fetal bovine serum; HA, hemagglutinin; COX, cyclooxygenase; CYP, cytochrome; HOX, homeobox. are associated with chromosomal rearrangements that lead to the expression of chimeric fusion proteins (1.Scandura J.M. Boccuni P. Cammenga J. Nimer S.D. Oncogene. 2002; 21: 3422-3444Crossref PubMed Scopus (86) Google Scholar). Genes encoding nuclear pore proteins (nucleoporins) are frequently rearranged in acute leukemia, particularly AML. Nup214 (CAN) was the first nucleoporin to be implicated in the pathogenesis of acute leukemia (2.Kraemer D. Wozniak R.W. Blobel G. Radu A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1519-1523Crossref PubMed Google Scholar). However, the gene encoding another nucleoporin, Nup98, has recently emerged as a much more frequent target of chromosomal rearrangements in leukemia. At least 17 different chromosomal rearrangements involving nucleoporin genes have been described in leukemia, 15 of which involve Nup98 (3.Lam D.H. Aplan P.D. Leukemia (Baltimore). 2001; 15: 1689-1695Crossref PubMed Google Scholar, 4.Jaju R.J. Fidler C. Haas O.A. Strickson A.J. Watkins F. Clark K. Cross N.C. Cheng J.F. Aplan P.D. Kearney L. Boultwood J. Wainscoat J.S. Blood. 2001; 98: 1264-1267Crossref PubMed Scopus (196) Google Scholar, 5.Fujino T. Suzuki A. Ito Y. Ohyashiki K. Hatano Y. Miura I. Nakamura T. Blood. 2002; 99: 1428-1433Crossref PubMed Scopus (62) Google Scholar, 6.Rosati R. La Starza R. Veronese A. Aventin A. Schwienbacher C. Vallespi T. Negrini M. Martelli M.F. Mecucci C. Blood. 2002; 99: 3857-3860Crossref PubMed Scopus (138) Google Scholar, 7.Suzuki A. Ito Y. Sashida G. Honda S. Katagiri T. Fujino T. Nakamura T. Ohyashiki K. Br. J. Haematol. 2002; 116: 170-172Crossref PubMed Scopus (23) Google Scholar, 8.Taketani T. Taki T. Shibuya N. Ito E. Kitazawa J. Terui K. Hayashi Y. Cancer Res. 2002; 62: 33-37PubMed Google Scholar, 9.Taketani T. Taki T. Shibuya N. Kikuchi A. Hanada R. Hayashi Y. Cancer Res. 2002; 62: 4571-4574PubMed Google Scholar, 10.Taketani T. Taki T. Ono R. Kobayashi Y. Ida K. Hayashi Y. Genes Chromosomes Cancer. 2002; 34: 437-443Crossref PubMed Scopus (45) Google Scholar, 11.Panagopoulos I. Isaksson M. Billstrom R. Strombeck B. Mitelman F. Johansson B. Genes Chromosomes Cancer. 2003; 36: 107-112Crossref PubMed Scopus (45) Google Scholar, 12.Lahortiga I. Vizmanos J.L. Agirre X. Vazquez I. Cigudosa J.C. Larrayoz M.J. Sala F. Gorosquieta A. Perez-Equiza K. Calasanz M.J. Odero M.D. Cancer Res. 2003; 63: 3079-3083PubMed Google Scholar). Nup98 gene rearrangements are more frequent in patients that had been previously treated with topoisomerase II inhibitors and may also be more common in Asian populations (3.Lam D.H. Aplan P.D. Leukemia (Baltimore). 2001; 15: 1689-1695Crossref PubMed Google Scholar, 4.Jaju R.J. Fidler C. Haas O.A. Strickson A.J. Watkins F. Clark K. Cross N.C. Cheng J.F. Aplan P.D. Kearney L. Boultwood J. Wainscoat J.S. Blood. 2001; 98: 1264-1267Crossref PubMed Scopus (196) Google Scholar, 5.Fujino T. Suzuki A. Ito Y. Ohyashiki K. Hatano Y. Miura I. Nakamura T. Blood. 2002; 99: 1428-1433Crossref PubMed Scopus (62) Google Scholar, 6.Rosati R. La Starza R. Veronese A. Aventin A. Schwienbacher C. Vallespi T. Negrini M. Martelli M.F. Mecucci C. Blood. 2002; 99: 3857-3860Crossref PubMed Scopus (138) Google Scholar, 7.Suzuki A. Ito Y. Sashida G. Honda S. Katagiri T. Fujino T. Nakamura T. Ohyashiki K. Br. J. Haematol. 2002; 116: 170-172Crossref PubMed Scopus (23) Google Scholar, 8.Taketani T. Taki T. Shibuya N. Ito E. Kitazawa J. Terui K. Hayashi Y. Cancer Res. 2002; 62: 33-37PubMed Google Scholar, 9.Taketani T. Taki T. Shibuya N. Kikuchi A. Hanada R. Hayashi Y. Cancer Res. 2002; 62: 4571-4574PubMed Google Scholar, 10.Taketani T. Taki T. Ono R. Kobayashi Y. Ida K. Hayashi Y. Genes Chromosomes Cancer. 2002; 34: 437-443Crossref PubMed Scopus (45) Google Scholar, 11.Panagopoulos I. Isaksson M. Billstrom R. Strombeck B. Mitelman F. Johansson B. Genes Chromosomes Cancer. 2003; 36: 107-112Crossref PubMed Scopus (45) Google Scholar, 13.La Starza R. Trubia M. Crescenzi B. Matteucci C. Negrini M. Martelli M.F. Pelicci P.G. Mecucci C. Genes Chromosomes Cancer. 2003; 36: 420-423Crossref PubMed Scopus (28) Google Scholar). Patients with nucleoporin gene rearrangements tend to be young, and their AML is usually refractory to therapy. Nucleoporin gene rearrangements result in the expression of chimeric proteins that consist of a portion of the nucleoporin fused to a portion of a partner protein. Both Nup98 and Nup214 belong to a subset of nucleoporins containing FG repeats (14.Corbett A.H. Silver P.A. Microbiol. Mol. Biol. Rev. 1997; 61: 193-211Crossref PubMed Scopus (169) Google Scholar, 15.Stoffler D. Fahrenkrog B. Aebi U. Curr. Opin. Cell Biol. 1999; 11: 391-401Crossref PubMed Scopus (292) Google Scholar). In all chromosomal rearrangements involving nucleoporins, the fusion product that is expressed in the leukemic cells contains these FG repeats (2.Kraemer D. Wozniak R.W. Blobel G. Radu A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1519-1523Crossref PubMed Google Scholar, 3.Lam D.H. Aplan P.D. Leukemia (Baltimore). 2001; 15: 1689-1695Crossref PubMed Google Scholar, 4.Jaju R.J. Fidler C. Haas O.A. Strickson A.J. Watkins F. Clark K. Cross N.C. Cheng J.F. Aplan P.D. Kearney L. Boultwood J. Wainscoat J.S. Blood. 2001; 98: 1264-1267Crossref PubMed Scopus (196) Google Scholar, 5.Fujino T. Suzuki A. Ito Y. Ohyashiki K. Hatano Y. Miura I. Nakamura T. Blood. 2002; 99: 1428-1433Crossref PubMed Scopus (62) Google Scholar, 6.Rosati R. La Starza R. Veronese A. Aventin A. Schwienbacher C. Vallespi T. Negrini M. Martelli M.F. Mecucci C. Blood. 2002; 99: 3857-3860Crossref PubMed Scopus (138) Google Scholar, 7.Suzuki A. Ito Y. Sashida G. Honda S. Katagiri T. Fujino T. Nakamura T. Ohyashiki K. Br. J. Haematol. 2002; 116: 170-172Crossref PubMed Scopus (23) Google Scholar, 8.Taketani T. Taki T. Shibuya N. Ito E. Kitazawa J. Terui K. Hayashi Y. Cancer Res. 2002; 62: 33-37PubMed Google Scholar, 9.Taketani T. Taki T. Shibuya N. Kikuchi A. Hanada R. Hayashi Y. Cancer Res. 2002; 62: 4571-4574PubMed Google Scholar, 10.Taketani T. Taki T. Ono R. Kobayashi Y. Ida K. Hayashi Y. Genes Chromosomes Cancer. 2002; 34: 437-443Crossref PubMed Scopus (45) Google Scholar, 11.Panagopoulos I. Isaksson M. Billstrom R. Strombeck B. Mitelman F. Johansson B. Genes Chromosomes Cancer. 2003; 36: 107-112Crossref PubMed Scopus (45) Google Scholar, 12.Lahortiga I. Vizmanos J.L. Agirre X. Vazquez I. Cigudosa J.C. Larrayoz M.J. Sala F. Gorosquieta A. Perez-Equiza K. Calasanz M.J. Odero M.D. Cancer Res. 2003; 63: 3079-3083PubMed Google Scholar, 13.La Starza R. Trubia M. Crescenzi B. Matteucci C. Negrini M. Martelli M.F. Pelicci P.G. Mecucci C. Genes Chromosomes Cancer. 2003; 36: 420-423Crossref PubMed Scopus (28) Google Scholar). In 8 of the 15 Nup98 gene rearrangements, the fusion partner is a transcription factor of the homeodomain family (2.Kraemer D. Wozniak R.W. Blobel G. Radu A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1519-1523Crossref PubMed Google Scholar, 3.Lam D.H. Aplan P.D. Leukemia (Baltimore). 2001; 15: 1689-1695Crossref PubMed Google Scholar, 4.Jaju R.J. Fidler C. Haas O.A. Strickson A.J. Watkins F. Clark K. Cross N.C. Cheng J.F. Aplan P.D. Kearney L. Boultwood J. Wainscoat J.S. Blood. 2001; 98: 1264-1267Crossref PubMed Scopus (196) Google Scholar, 5.Fujino T. Suzuki A. Ito Y. Ohyashiki K. Hatano Y. Miura I. Nakamura T. Blood. 2002; 99: 1428-1433Crossref PubMed Scopus (62) Google Scholar, 6.Rosati R. La Starza R. Veronese A. Aventin A. Schwienbacher C. Vallespi T. Negrini M. Martelli M.F. Mecucci C. Blood. 2002; 99: 3857-3860Crossref PubMed Scopus (138) Google Scholar, 7.Suzuki A. Ito Y. Sashida G. Honda S. Katagiri T. Fujino T. Nakamura T. Ohyashiki K. Br. J. Haematol. 2002; 116: 170-172Crossref PubMed Scopus (23) Google Scholar, 8.Taketani T. Taki T. Shibuya N. Ito E. Kitazawa J. Terui K. Hayashi Y. Cancer Res. 2002; 62: 33-37PubMed Google Scholar, 9.Taketani T. Taki T. Shibuya N. Kikuchi A. Hanada R. Hayashi Y. Cancer Res. 2002; 62: 4571-4574PubMed Google Scholar, 10.Taketani T. Taki T. Ono R. Kobayashi Y. Ida K. Hayashi Y. Genes Chromosomes Cancer. 2002; 34: 437-443Crossref PubMed Scopus (45) Google Scholar, 11.Panagopoulos I. Isaksson M. Billstrom R. Strombeck B. Mitelman F. Johansson B. Genes Chromosomes Cancer. 2003; 36: 107-112Crossref PubMed Scopus (45) Google Scholar, 12.Lahortiga I. Vizmanos J.L. Agirre X. Vazquez I. Cigudosa J.C. Larrayoz M.J. Sala F. Gorosquieta A. Perez-Equiza K. Calasanz M.J. Odero M.D. Cancer Res. 2003; 63: 3079-3083PubMed Google Scholar, 13.La Starza R. Trubia M. Crescenzi B. Matteucci C. Negrini M. Martelli M.F. Pelicci P.G. Mecucci C. Genes Chromosomes Cancer. 2003; 36: 420-423Crossref PubMed Scopus (28) Google Scholar). The prototype of these fusion proteins is Nup98-HOXA9, which results from t(7;11)(p15;p15) and consists of the N-terminal FG repeat region of Nup98 fused to the C terminus of HOXA9 that includes the DNA binding homeodomain (Fig. 1). Nup98 is present both at the nuclear pore complex and within the nuclear interior (16.Griffis E.R. Altan N. Lippincott-Schwartz J. Powers M.A. Mol. Biol. Cell. 2002; 13: 1282-1297Crossref PubMed Scopus (192) Google Scholar, 17.Fontoura B.M. Dales S. Blobel G. Zhong H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3208-3213Crossref PubMed Scopus (78) Google Scholar, 18.Powers M.A. Macaulay C. Masiarz F.R. Forbes D.J. J. Cell Biol. 1995; 128: 721-736Crossref PubMed Scopus (104) Google Scholar, 19.Radu A. Moore M.S. Blobel G. Cell. 1995; 81: 215-222Abstract Full Text PDF PubMed Google Scholar). There is evidence that the FG repeat region of Nup98 interacts with a number of molecules involved in protein import including karyopherins β1, β2, and β3 and RCC1 (regulator of chromatin condensation 1) and that it plays a role in nuclear import (19.Radu A. Moore M.S. Blobel G. Cell. 1995; 81: 215-222Abstract Full Text PDF PubMed Google Scholar, 20.Yaseen N.R. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4451-4456Crossref PubMed Scopus (112) Google Scholar, 21.Fontoura B.M. Blobel G. Yaseen N.R. J. Biol. Chem. 2000; 275: 31289-31296Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). In addition, Nup98 interacts with the RNA export proteins RAE1 and TAP, and there is evidence that it functions in RNA export from the nucleus (3.Lam D.H. Aplan P.D. Leukemia (Baltimore). 2001; 15: 1689-1695Crossref PubMed Google Scholar, 22.Wang X. Babu J.R. Harden J.M. Jablonski S.A. Gazi M.H. Lingle W.L. de Groen P.C. Yen T.J. van Deursen J.M. J. Biol. Chem. 2001; 276: 26559-26567Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 23.Blevins M.B. Smith A.M. Phillips E.M. Powers M.A. J. Biol. Chem. 2003; 278: 20979-20988Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 24.Bachi A. Braun I.C. Rodrigues J.P. Pante N. Ribbeck K. von Kobbe C. Kutay U. Wilm M. Gorlich D. Carmo-Fonseca M. Izaurralde E. RNA (N. Y.). 2000; 6: 136-158Crossref PubMed Scopus (264) Google Scholar, 25.Powers M.A. Forbes D.J. Dahlberg J.E. Lund E. J. Cell Biol. 1997; 136: 241-250Crossref PubMed Scopus (169) Google Scholar). The function of Nup98 within the nucleus is not entirely clear. It has been reported that Nup98 is present on fibrillar structures inside the nucleus, suggesting a role in intranuclear trafficking (17.Fontoura B.M. Dales S. Blobel G. Zhong H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3208-3213Crossref PubMed Scopus (78) Google Scholar). Other studies suggest that intranuclear Nup98 may be involved in the trafficking of RNA from transcription sites to the nuclear pore complex (16.Griffis E.R. Altan N. Lippincott-Schwartz J. Powers M.A. Mol. Biol. Cell. 2002; 13: 1282-1297Crossref PubMed Scopus (192) Google Scholar). Finally, there is evidence that the FG repeat region of Nup98 can activate transcription from a GAL4-responsive promoter when attached to a GAL4 DNA binding domain, suggesting a possible role for Nup98 in transcription (26.Kasper L.H. Brindle P.K. Schnabel C.A. Pritchard C.E. Cleary M.L. van Deursen J.M. Mol. Cell. Biol. 1999; 19: 764-776Crossref PubMed Google Scholar). HOXA9 is one of a large family of transcription factors characterized by the presence of a DNA binding homeodomain (27.Alberts B. Johnson A. Lewis J. Raff M. Roberts K. Walter P. Molecular Biology of the Cell. 4th Ed. Garland Science, New York2002: 1157-1258Google Scholar). Homeodomain transcription factors are involved in patterning the anteroposterior axis of the body during embryonic development (27.Alberts B. Johnson A. Lewis J. Raff M. Roberts K. Walter P. Molecular Biology of the Cell. 4th Ed. Garland Science, New York2002: 1157-1258Google Scholar) and play important and complex roles during hematopoiesis (28.Payne K.J. Crooks G.M. Immunol. Rev. 2002; 187: 48-64Crossref PubMed Scopus (29) Google Scholar, 29.Chiba S. Int. J. Hematol. 1998; 68: 343-353Crossref PubMed Google Scholar, 30.Owens B.M. Hawley R.G. Stem Cells. 2002; 20: 364-379Crossref PubMed Google Scholar, 31.Lawrence H.J. Sauvageau G. Humphries R.K. Largman C. Stem Cells. 1996; 14: 281-291Crossref PubMed Google Scholar). There are two major groups of homeodomain proteins. Class I includes the HOX genes that exist in 4 genomic clusters, designated A-D, on chromosomes 7, 17, 12, and 2, respectively, and are divided into 13 paralogous groups numbered 1–13 (30.Owens B.M. Hawley R.G. Stem Cells. 2002; 20: 364-379Crossref PubMed Google Scholar). Most of the homeodomain fusion partners of Nup98, including HOXA9, belong to the so-called Abd-B group of HOX genes that includes paralogous groups 9–13 (2.Kraemer D. Wozniak R.W. Blobel G. Radu A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1519-1523Crossref PubMed Google Scholar, 3.Lam D.H. Aplan P.D. Leukemia (Baltimore). 2001; 15: 1689-1695Crossref PubMed Google Scholar, 4.Jaju R.J. Fidler C. Haas O.A. Strickson A.J. Watkins F. Clark K. Cross N.C. Cheng J.F. Aplan P.D. Kearney L. Boultwood J. Wainscoat J.S. Blood. 2001; 98: 1264-1267Crossref PubMed Scopus (196) Google Scholar, 5.Fujino T. Suzuki A. Ito Y. Ohyashiki K. Hatano Y. Miura I. Nakamura T. Blood. 2002; 99: 1428-1433Crossref PubMed Scopus (62) Google Scholar, 6.Rosati R. La Starza R. Veronese A. Aventin A. Schwienbacher C. Vallespi T. Negrini M. Martelli M.F. Mecucci C. Blood. 2002; 99: 3857-3860Crossref PubMed Scopus (138) Google Scholar, 7.Suzuki A. Ito Y. Sashida G. Honda S. Katagiri T. Fujino T. Nakamura T. Ohyashiki K. Br. J. Haematol. 2002; 116: 170-172Crossref PubMed Scopus (23) Google Scholar, 8.Taketani T. Taki T. Shibuya N. Ito E. Kitazawa J. Terui K. Hayashi Y. Cancer Res. 2002; 62: 33-37PubMed Google Scholar, 9.Taketani T. Taki T. Shibuya N. Kikuchi A. Hanada R. Hayashi Y. Cancer Res. 2002; 62: 4571-4574PubMed Google Scholar, 10.Taketani T. Taki T. Ono R. Kobayashi Y. Ida K. Hayashi Y. Genes Chromosomes Cancer. 2002; 34: 437-443Crossref PubMed Scopus (45) Google Scholar, 11.Panagopoulos I. Isaksson M. Billstrom R. Strombeck B. Mitelman F. Johansson B. Genes Chromosomes Cancer. 2003; 36: 107-112Crossref PubMed Scopus (45) Google Scholar, 12.Lahortiga I. Vizmanos J.L. Agirre X. Vazquez I. Cigudosa J.C. Larrayoz M.J. Sala F. Gorosquieta A. Perez-Equiza K. Calasanz M.J. Odero M.D. Cancer Res. 2003; 63: 3079-3083PubMed Google Scholar, 13.La Starza R. Trubia M. Crescenzi B. Matteucci C. Negrini M. Martelli M.F. Pelicci P.G. Mecucci C. Genes Chromosomes Cancer. 2003; 36: 420-423Crossref PubMed Scopus (28) Google Scholar). Class II includes genes such as PMX1, PBX1, and MEIS1, that are scattered throughout the genome (30.Owens B.M. Hawley R.G. Stem Cells. 2002; 20: 364-379Crossref PubMed Google Scholar). HOXA9 has been shown to bind DNA cooperatively with PBX1 and/or MEIS1 (32.Shen W.F. Rozenfeld S. Kwong A. Kom ves L.G. Lawrence H.J. Largman C. Mol. Cell. Biol. 1999; 19: 3051-3061Crossref PubMed Google Scholar, 33.Shen W.F. Montgomery J.C. Rozenfeld S. Moskow J.J. Lawrence H.J. Buchberg A.M. Largman C. Mol. Cell. Biol. 1997; 17: 6448-6458Crossref PubMed Google Scholar). Although the normal functions of HOXA9 are not well understood, there is ample evidence that it is involved in the pathogenesis of AML, particularly in collaboration with MEIS1. HOXA9 overexpression can immortalize myeloid progenitors in vitro and inhibit some of their differentiation pathways (34.Calvo K.R. Sykes D.B. Pasillas M. Kamps M.P. Mol. Cell. Biol. 2000; 20: 3274-3285Crossref PubMed Scopus (111) Google Scholar). When mice are transplanted with bone marrow cells overexpressing HOXA9, they develop AML in an average of 128 days, a period that is shortened to 57 days by MEIS1 coexpression (35.Kroon E. Thorsteinsdottir U. Mayotte N. Nakamura T. Sauvageau G. EMBO J. 2001; 20: 350-361Crossref PubMed Scopus (174) Google Scholar). Furthermore, HOXA9 and MEIS1 are frequently coexpressed in human AML (36.Lawrence H.J. Rozenfeld S. Cruz C. Matsukuma K. Kwong A. Komuves L. Buchberg A.M. Largman C. Leukemia (Baltimore). 1999; 13: 1993-1999Crossref PubMed Google Scholar). The Nup98-HOXA9 fusion results in replacement of the transcriptional regulatory region of HOXA9 by the FG repeat region of Nup98 (Fig. 1). In contrast to wild-type Nup98, the Nup98-HOXA9 chimera is primarily intranuclear (26.Kasper L.H. Brindle P.K. Schnabel C.A. Pritchard C.E. Cleary M.L. van Deursen J.M. Mol. Cell. Biol. 1999; 19: 764-776Crossref PubMed Google Scholar). Nup98-HOXA9 increases the proliferative capacity of bone marrow progenitors (35.Kroon E. Thorsteinsdottir U. Mayotte N. Nakamura T. Sauvageau G. EMBO J. 2001; 20: 350-361Crossref PubMed Scopus (174) Google Scholar, 37.Calvo K.R. Sykes D.B. Pasillas M.P. Kamps M.P. Oncogene. 2002; 21: 4247-4256Crossref PubMed Scopus (93) Google Scholar). Transplanting mice with hematopoietic stem cells that express Nup98-HOXA9 induces a myeloproliferative disease with development of AML within an average of 230 days (35.Kroon E. Thorsteinsdottir U. Mayotte N. Nakamura T. Sauvageau G. EMBO J. 2001; 20: 350-361Crossref PubMed Scopus (174) Google Scholar). Coexpression of Nup98-HOXA9 with MEIS1 shortens the period of AML development to an average of 142 days (35.Kroon E. Thorsteinsdottir U. Mayotte N. Nakamura T. Sauvageau G. EMBO J. 2001; 20: 350-361Crossref PubMed Scopus (174) Google Scholar). Coexpression of Nup98-HOXA9 with the Bcr-Abl fusion oncoprotein reduces the period to AML even further, to 21 days (38.Dash A.B. Williams I.R. Kutok J.L. Tomasson M.H. Anastasiadou E. Lindahl K. Li S. Van Etten R.A. Borrow J. Housman D. Druker B. Gilliland D.G. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7622-7627Crossref PubMed Scopus (160) Google Scholar). Thus, it is clear that Nup98-HOXA9 plays a causative role in the development of AML, although additional factors may be necessary for rapid induction of a full-blown AML phenotype. The mechanisms by which Nup98-HOXA9 and other Nup98 fusions contribute to the pathogenesis of AML are not well understood. In this study we provide strong evidence that Nup98-HOXA9 acts as an aberrant transcription factor in myeloid cells and identify 102 target genes. Of these, 92 are up-regulated, whereas only 10 are down-regulated, indicating that Nup98-HOXA9 acts primarily as a transcriptional activator. In contrast, wild-type HOXA9 has only 13 target genes, and Nup98 has none. Several of the genes induced by Nup98-HOXA9 are associated with increased cell proliferation and survival, suggesting possible pathways involved in leukemogenesis. Interestingly, the gene encoding a drug-metabolizing enzyme, CYP3A5, is markedly induced by Nup98-HOXA9 and may explain some aspects of the epidemiology of Nup98-HOXA9-associated AML, particularly its association with etoposide treatment and its incidence in Asian populations. Cell Culture—K562 cells (ATCC, CCL-243) were cultured in Iscove's modified Dulbecco's medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Invitrogen), 2 mm l-glutamine, and 100 units/ml penicillin/streptomycin (Invitrogen). GP-293 packaging cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% heat-inactivated FBS, 2 mm l-glutamine, and 100 units/ml penicillin/streptomycin. All cells were maintained at 37 °C, 5% CO2. Plasmid Constructs—Nup98-HoxA9 and full-length Nup98 cDNAs with N-terminal HA tags in pUHD10S vector (39.Porter C.M. Clipstone N.A. J. Immunol. 2002; 168: 4936-4945Crossref PubMed Google Scholar) were a kind gift from Dr. Jan van Deursen (Mayo Clinic). The cDNAs were subcloned into the MSCV-IRES-GFP retroviral expression vector, kindly provided by Dr. Neil Clipstone (Northwestern University) (39.Porter C.M. Clipstone N.A. J. Immunol. 2002; 168: 4936-4945Crossref PubMed Google Scholar). Full-length Nup98 was digested out of pUHD10S with EcoR1 and ligated into the EcoRI site of MSCV-IRES-GFP. Nup98-HoxA9 was digested out of puHD10s with EcoRI and XbaI (New England Biolabs) followed by filling in the recessed ends with Klenow and ligating into the HpaI site of MSCV-IRES-GFP. Wild-type HA-tagged human HOXA9 was constructed by PCR, checked by sequencing, and similarly subcloned into the HpaI site of MSCV-IRES-GFP. The Nup98, HOXA9, and Nup98-HOXA9 cDNAs were also subcloned into the pcDNA3 vector (Invitrogen) using the EcoRI (Nup98) and EcoRI-XbaI (HOXA9 and Nup98-HOXA9) sites. Retrovirus Production and Infection—The retroviral expression vectors were cotransfected with the vesicular stomatitis virus glycoprotein plasmid (pVSV-G) into the GP-293 packaging cell line using the LipofectAMINE Plus reagent (Invitrogen). Plasmid-containing medium was removed 24 h post-transfection and replaced with Dulbecco's modified Eagle's medium supplemented with 10% FBS, and the cells were incubated at 32 °C. Virus-containing supernatant was then collected 48 h post-transfection and stored at –80 °C before use. K562 cells were suspended in viral supernatant at 2 × 105 cells/ml in the presence of 8 μg/ml hexadimethrine bromide (Sigma). The cells were seeded in a 24-well plate and subjected to spinoculation at 500 × g for 2 h at room temperature followed by replacement of the viral supernatant with Iscove's modified Dulbecco's medium supplemented with 10% FBS. Green fluorescent protein (GFP)-expressing cells were then sorted 48 h post-infection using a Beckman Coulter Elite ESP cell sorter. Western Blot Analysis—GFP-expressing cells were lysed in SDS-PAGE sample buffer 48 h after sorting, and the protein equivalent of 1 × 105 cells was subjected to SDS-PAGE electrophoresis. The respective proteins were detected using the anti-HA antibody 12CA5 (Roche Applied Science). RNA Isolation and Oligonucleotide Array Expression Analysis—Cytoplasmic RNA was isolated from K562 cells 48 h post-sorting using the RNeasy mini kit (Qiagen). Total RNA quality control was performed by running 25–50 ng on an RNA 6000 Nano Assay (Agilent) using a Bioanalyzer 2100. For labeling, 2 μg of good quality total RNA was reverse-transcribed with an oligo-dT-T7 (Genset), and double-stranded cDNA was generated with the superscript double-stranded cDNA synthesis custom kit (Invitrogen). In an in vitro transcription step with T7 RNA polymerase (MessageAmp aRNA kit from Ambion), the cDNA was linearly amplified and labeled with biotinylated nucleotides (Enzo Diagnostics). Ten μg of labeled and fragmented cRNA was then hybridized onto a human genome U133A expression array (HG-U133A) (Affymetrix) for 16 h at 45 °C. Post-hybridization staining and washing were performed according to the manufacturer's instructions. Finally, chips were scanned with a Hewlett Packard argon-ion laser confocal scanner at the Microarray Facility of the Memorial-Sloan Kettering Cancer Center. Image and Data Analysis—The microarray image data were first quantitated using MAS 5.0 (microarray suite) with the default parameters for the statistical algorithm and all probe set scaling with a target intensity of 500. The data were then filtered so that both the absolute value of the fold change was greater than or equal to 2, and the change in p value was less than or equal to 0.001. Additionally we removed genes that were scored as increasing and also scored as absent (A) in the numerator (i.e. the experiment in the ratio whose value is the numerator) and genes scored as decreasing and scored as absent in the denominator (also referred to as the base-line experiment). Experiments were done in replicate, and we chose a conservative procedure for combining the replicate data. We intersected the filtered list for each replicate given a list of genes that passed the filtered criteria for all of the replicates. Real-time PCR—Real-time PCR was performed on cDNA synthesized from RNA isolated from K562 cells infected with either empty MSCV-IRES-GFP or Nup98/HOXA9 retroviral constructs using the iCycler iQ real-time PCR detection system (Bio-Rad). The iCycler was formatted for 96-well plates containing 25-μl PCR reactions. PCR master mixes were made such that each 25-μl reaction contained 12.5 μl of iQ Sybr Green Supermix (Bio-Rad), 2 μl of cDNA template (diluted 1:200), and 0.8 μm sense and antisense gene primers. PCR conditions consisted of an initial denaturing step for 5 min at 95 °C followed by 40 cycles of a 3-segment step consisting of denaturation for 30 s at 95 °C, annealing for 30 s at 55 °C, and elongation for 30 s at 72 °C. Real-time analysis was taken during the 30-s annealing step. After real-time analysis, a melting curve was established for all samples to ensure specific amplification. Gene quantification was determined based upon a relative standard curve from a dilution series of the template cDNA (1:20, 1:200,1:2000, and 1:20000). A negative control where no template DNA was used was run on each plate as well as a comparison of glyceraldehyde phosphate dehydrogenase between the MSCV-IRES-GFP and Nup98/HOXA9 samples. Glyceraldehyde phosphate dehydrogenase served to equilibrate the starting material between the two experimental conditions. All unknown samples as well as controls were run in triplicate on the same plate (except for the negative control). Data analysis was performed using the iCycler iQ Optical System Software Version 3.0a (Bio-Rad). Transfection of K562 Cells and Luciferase Assay—K562 cells were transfected with a 10 μg of the pXP1/cyclooxygenase-1 (COX-1) construc" @default.
• W2003706637 created "2016-06-24" @default.
• W2003706637 creator A5007696387 @default.
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• W2003706637 creator A5091124074 @default.
• W2003706637 date "2004-01-01" @default.
• W2003706637 modified "2023-10-14" @default.
• W2003706637 title "The Oncogene Nup98-HOXA9 Induces Gene Transcription in Myeloid Cells" @default.
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