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Expression of PAX3 in Ewing's Sarcoma Family of Tumors
BIOCHEMICAL AND MOLECULAR MEDICINE ARTICLE NO.
60, 121–126 (1997)
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Expression of PAX3 in Ewing’s Sarcoma Family of Tumors THEODOR W. SCHULTE,1 JEFFREY A. TORETSKY,* ELISABETH RESS, LEE HELMAN,* AND LEONARD M. NECKERS Clinical Pharmacology Branch and *Pediatric Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892 Received October 9, 1996
PAX3 is a member of the paired box family of genes (Pax genes) which were first described in Drosophila (9). Because of its high expression in the neural crest during embryogenesis, we investigated PAX3 expression in neural crest-derived ES/PNET cell lines and tumor specimens. Our data demonstrate expression of PAX3 in the majority but not in all of these specimens.
The Ewing’s sarcoma family of tumors (ESFT) is the second most common pediatric malignancy originating in the bone and is characterized by the t(11; 22) translocation. PAX3, a member of the paired box family of genes, is expressed during embryonal development of neural crest cells and is involved in the t(2; 13) translocation found in alveolar rhabdomyosarcoma. Since ESFTs are believed to be derived from neural crest tissue, we screened a series of Ewing’s sarcoma and peripheral neuroectodermal tumor cell lines and tumor specimens for expression of PAX3. We found expression of PAX3 in most, but not all, of the specimens analyzed, including cell lines and patient material. q 1997 Academic Press
MATERIAL AND METHODS Cell lines and tissue preparations. The cell line JR0614 was derived from a tumor explant (metastatic PNET) (10). The PNET cell lines TC32 and CHP100 were grown in RMPI 1640 as previously published (11,12). The cell lines RDES (ES), HeLa (epithelioid cervical carcinoma), PC3 (prostate adenocarcinoma), Jurkat (acute T-cell leukemia), IMR32 (neuroblastoma), DAOY (medulloblastoma), D 23 8 ( m ed u ll o bl a s to m a) , U 9 37 ( h is t io c y ti c lymphoma), U87 (glioblastoma), and K562 (chronic myelogenous leukemia) were purchased from ATCC. MCF7 cells were obtained from Dr. Kenneth Cowan (NCI) and maintained in Dulbecco’s modified Eagle’s medium containing 10% bovine calf serum and 10 mM Hepes. Tumor tissue was obtained from patients at the time of surgery and snap frozen at 0707C. Informed consent was obtained prior to surgery for the collection of tissue.
Ewing’s sarcoma family of tumors (ESFTs) consists of the ES of bone, extraosseous Ewing’s sarcoma (ES), and peripheral neuroectodermal tumor (PNET) and is characterized by the t(11; 22) (q24; q12) translocation (1–3). ES and PNET are thought to be of neuroectodermal origin because they express neuroectoderm-associated antigens (such as ganglioside GD-2, N-CAM, HNK-1 antigen (4,5)) and because ES cells undergo neuronal differentiation when treated with cyclic adenosine monophosphate or phorbol myristic acid (6). Further, these tumors typically express high levels of choline acetyltransferase and low or undetectable levels of adrenergic enzymes, a characteristic of parasympathetic neurons (7,8).
RNA isolation and RT–PCR. Total RNA was prepared from cultured cells growing in log phase, or from tumor tissue, using RNazol B according to the manufacturer’s recommendations (Tel-Test, Inc.,
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To whom correspondence should be addressed at Clinical Pharmacology Branch, Building 10, Room 13N240, 10 Center Dr. MSC 1928, Bethesda, MD 20892-1928. Fax: (301) 402-0575. Email: [email protected]. 121
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FIG. 1. Expression of PAX3 in various cell lines. (A) 1 mg of total RNA from cells growing in log phase was reverse transcribed and amplified by PCR using PAX3- and b-actin-specific primers. (B) 20 mg of total protein from ESFT cell lines and HeLa and protein produced by in vitro translation of a Pax3 cDNA were analyzed by Western blotting. (C) RDES cells and MCF7 cells were grown on coverslips and stained for PAX3 protein by immunofluorescence. The DNA intercalating dye Hoechst 33258 was used to identify cell nuclei.
Friendswood, TX). One microgram of total RNA was reverse transcribed using random hexamers (Gene Amp RNA PCR kit, Perkin–Elmer Cetus, Foster City, CA). PCR was performed with the upstream primer PP1, 5*-acctcagtcagatgaaggctccgat-3* (exon 5, PAX3), and the downstream primer PP2, 5*-tgaggtctgtggacggtgctactg-3* (exon 7, PAX3). Amplifica-
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tion was performed for 35 cycles at 957C for 1 min, 657C for 1 min, and 727C for 3 min. Twenty percent of the reaction mixture was analyzed on a 2% agarose gel and transferred to Nytran Maximum Strength membrane (Schleicher & Schuell, Keene, NH). Membranes were probed with the end-labeled oligonucleotide PP3, 5*-ACCTCAGTCAGATGAAG-
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FIG. 1—Continued
GCTCCGAT-3*, which corresponds to a region in exon 5 of PAX3. In order to control for the quality of the RNA preparation and transcription, RT–PCR was performed in parallel with b-actin primers. Experiments were done twice and negative controls were included in every PCR. Western blot analysis. Cell lines were rinsed in phosphate-buffered saline (PBS) and lysed in RIPA buffer (10 mM Tris–HCl, pH 7.2, 0.5% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 1 mM EDTA, 1 mM sodium orthovanadate) containing protease inhibitors (aprotinin 20 mg/ml, leupeptin 20 mg/ml, 1 mM PMSF). Lysates were centrifuged at 14,000g for 20 min at 47C. The protein concentration was measured using the BCA protein assay kit (Pierce, Rockford, IL). Twenty micrograms of total protein was analyzed on an 8% SDS–PAGE minigel as previously described (13). We used a PAX3 rabbit antiserum (14) as the primary antibody. As a posi-
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tive control murine Pax3 was in vitro translated from its full-length cDNA (14) using a rabbit reticulocyte lysate system (Promega, Madison, WI), and an aliquot of this preparation was included in Western blots. The murine Pax3 protein is over 98% homologous with human PAX3 and is recognized by the antibody we used (14). The experiment was performed twice and showed similar results. Immunofluorescence. RDES or MCF7 cells were grown on coverslips. PAX3 was visualized by immunofluorescence as previously described (15) using PAX3 polyclonal rabbit antibody (14). Hoechst 33258 (Molecular Probes, Eugene, OR) was used to visualize cell nuclei. RESULTS We first analyzed various cell lines for PAX3 expression using RT–PCR with PAX3-specific primers
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FIG. 2. Expression of PAX3 in ESFT patient material. RNA was prepared from frozen samples of tumor tissue from five patients with ES and six patients with PNET and used for RT–PCR with PAX3- and b-actin-specific primers. The specificity of the PAX3 PCR product was confirmed by hybridization with a radiolabeled probe.
(Fig. 1A). The specificity of the PCR product was confirmed by hybridization with a specific radiolabeled oligonucleotide probe (data not shown). In order to exclude the amplification of genomic DNA, primers were chosen from exons 5 and 7. PCR amplification in the absence of reverse transcriptase did not produce any detectable signal (data not shown). Several ESFT cell lines were found to express PAX3, including TC32, JR0614, and CHP100. PAX3 was also expressed in cell lines derived from a glioblastoma (U87) and a medulloblastoma (DAOY). A second medulloblastoma (D283) and a neuroblastoma cell line (IMR32) were negative, as were all cell lines derived from hematogenous (Jurkat, K562), lymphomatous (U937), and carcinomatous (Hela, PC3M) tissue (Fig. 1A). Next, we assessed PAX3 protein expression in the ESFT cell lines by Western blotting (Fig. 1B). RDES (ES), TC32 (PNET), and JR0614 (ES) gave a strong signal, while CHP100 expressed low but detectable amounts of PAX3 protein. As a negative control, HeLa cells did not express PAX3 by Western blot analysis. In vitro translated murine Pax3 was used as a positive control (see lane 1 in Fig. 1B). Immunofluorescence analysis of PAX3 expression in RDES cells (Fig. 1C) demonstrated the expected intranuclear localization of PAX3 protein. MCF7 cells served as a negative control. RNA was also prepared from ESFT tumor specimens and used for RT–PCR with PAX3-specific primers. Four of the five tumor specimens (80%) from patients with ES and three of the six tumor specimens (50%) from patients with PNET were found to express PAX3 mRNA (Fig. 2). The specific-
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ity of the PCR product was confirmed by hybridization with a radiolabeled probe. DISCUSSION ES and PNET are thought to be derived from cells of the neural crest (16). PAX3 is expressed in the neural crest during embryogenesis (17,18), as well as in in vitro models of neural crest differentiation (19,20). Since expression of the renal developmental genes PAX2 and PAX8 had been described in Wilms tumor (21–23), we hypothesized that PAX3 could be expressed in ES and PNET. We screened several ES and PNET cell lines and tumor specimens for expression of this paired box gene. We found PAX3 to be expressed in 4/4 ESFT cell lines and 7/11 ESFT tumor specimens. While PAX3 is expressed in a variety of tissues during early embryonal development, including mesencephalon, rhombencephalon, cerebellum, neural tube, neural crest, dermomyotome, and somites (24), expression is very limited in the adult organism (25). For this reason, expression of PAX3 in neuroectodermal tumors is noteworthy. It is impossible to compare the gene expression in ES cells to a normal cell of origin because such a cell has not yet been described (16). PAX genes have been considered protooncogenes because of the expression of gene family members in various tumors and because of certain functional properties of PAX genes. Specifically, high levels of PAX2 and PAX8 have been found in Wilms tumor and renal cell carcinoma (21–23,26), PAX5 was reported in the majority of cases analyzed in medul-
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loblastoma (27) and astrocytoma (28), while PAX8 was expressed in thyroid carcinoma (26,29). Aberrant PAX3 expression has also been implicated in the etiology of alveolar rhabdomyosarcoma, where a characteristic t(2; 13) chromosomal translocation leads to the formation of a hybrid protein that combines the DNA binding domains of PAX3 with the C-terminal activation domain of FKHR, a member of the forkhead family of transcription factors (30,31). Less frequently, the variant t(1; 13) leads to a PAX7/ FKHR fusion in alveolar rhabdomyosarcoma (32). Besides, PAX genes possess functional features that relate to oncogenesis. Data on PAX2 suggest a role of this gene in proliferation of kidney cells (33). The gene is downregulated shortly before or concurrent with terminal differentiation and is found reactivated in regenerating proximal tubule cells. PAX5 was shown to mediate loss of p53 function through transcriptional repression (34). Transfected Pax3 inhibits differentiation in cultured myoblasts (35). And finally, PAX genes were found to transform fibroblasts both in cell culture and in nude mice (36). Although our data suggest that PAX3 expression may be common in ESFT, we observed variability in the level of PAX3 expressed in the different ESFT cell lines, as well as lack of PAX3 expression in several ESFT patient samples. In addition, PAX3 expression is not restricted to this family of tumor, as we were able to demonstrate PAX3 expression in one of two medulloblastoma cell lines examined, as well as in the only glioblastoma cell line studied. Interestingly, however, no PAX3 expression was observed in several carcinoma-, leukemia-, and lymphoma-derived cell lines. At this point the functional significance of PAX3 expression in ESFT cells is not clear. PAX3 could be a lineage marker associated with certain cell types. But given the role PAX3 plays in development, and PAX family genes play in other forms of cancer (27,33,37,38), PAX3 expression may well be involved in oncogenesis. We believe that our findings warrant further study of the expression and function of PAX3 in ESFTs. ACKNOWLEDGMENTS We gratefully thank Dr. David Shapiro (St. Jude Research Hospital, Memphis, TN) for PAX3 antiserum and Pax3 cDNA. This project was supported by the Cooperative Human Tissue Network, which is funded by the National Cancer Institute.
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16. Loizaga JM. What’s new in Ewing’s tumor? Pathol Res Pract 189:616–620, 1993. 17. Goulding MD, Chalepakis G, Deutsch U, Erselius JR, Gruss P. Pax-3, a novel murine DNA binding protein expressed during early neurogenesis. EMBO J 10:1135–1147, 1991. 18. Chalepakis G, Goulding M, Read A, Strachan T, Gruss P. Molecular basis of splotch and Waardenburg Pax-3 mutations. Proc Natl Acad Sci USA 91:3685–3689, 1994. 19. Pruitt SC. Expression of Pax-3- and neuroectoderm-inducing activities during differentiation of P19 embryonal carcinoma cells. Development 116:573–583, 1992. 20. Pruitt SC. Discrete endogenous signals mediate neural competence and induction in P19 embryonal carcinoma stem cells. Development 120:3301–3312, 1994. 21. Dressler GR, Douglass EC. Pax-2 is a DNA-binding protein expressed in embryonic kidney and Wilms tumor. Proc Natl Acad Sci USA 89:1179–1183, 1992. 22. Gnarra JR, Dressler GR. Expression of Pax-2 in human renal cell carcinoma and growth inhibition by antisense oligonucleotides. Cancer Res 55:4092–4098, 1995. 23. Eccles MR, Yun K, Reeve AE, Fidler AE. Comparative in situ hybridization analysis of PAX2, PAX8, and WT1 gene transcription in human fetal kidney and Wilms’ tumors. Am J Pathol 146:40–45, 1995. 24. Tremblay P, Gruss P. Pax: Genes for mice and men. Pharmacol Ther 61:205–226, 1994. 25. Stoykova A, Gruss P. Roles of Pax-genes in developing and adult brain as suggested by expression patterns. J Neurosci 14:1395–1412, 1994. 26. Poleev A, Wendler F, Fickenscher H, Zannini MS, Yaginuma K, Abbott C, Plachov D. Distinct functional properties of three human paired-box-protein, PAX8, isoforms generated by alternative splicing in thyroid, kidney and Wilms’ tumors. Eur J Biochem 228:899–911, 1995. 27. Kozmik Z, Sure U, Ruedi D, Busslinger M, Aguzzi A. Deregu-
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Expression of PAX3 in Ewing’s Sarcoma Family of Tumors THEODOR W. SCHULTE,1 JEFFREY A. TORETSKY,* ELISABETH RESS, LEE HELMAN,* AND LEONARD M. NECKERS Clinical Pharmacology Branch and *Pediatric Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892 Received October 9, 1996
PAX3 is a member of the paired box family of genes (Pax genes) which were first described in Drosophila (9). Because of its high expression in the neural crest during embryogenesis, we investigated PAX3 expression in neural crest-derived ES/PNET cell lines and tumor specimens. Our data demonstrate expression of PAX3 in the majority but not in all of these specimens.
The Ewing’s sarcoma family of tumors (ESFT) is the second most common pediatric malignancy originating in the bone and is characterized by the t(11; 22) translocation. PAX3, a member of the paired box family of genes, is expressed during embryonal development of neural crest cells and is involved in the t(2; 13) translocation found in alveolar rhabdomyosarcoma. Since ESFTs are believed to be derived from neural crest tissue, we screened a series of Ewing’s sarcoma and peripheral neuroectodermal tumor cell lines and tumor specimens for expression of PAX3. We found expression of PAX3 in most, but not all, of the specimens analyzed, including cell lines and patient material. q 1997 Academic Press
MATERIAL AND METHODS Cell lines and tissue preparations. The cell line JR0614 was derived from a tumor explant (metastatic PNET) (10). The PNET cell lines TC32 and CHP100 were grown in RMPI 1640 as previously published (11,12). The cell lines RDES (ES), HeLa (epithelioid cervical carcinoma), PC3 (prostate adenocarcinoma), Jurkat (acute T-cell leukemia), IMR32 (neuroblastoma), DAOY (medulloblastoma), D 23 8 ( m ed u ll o bl a s to m a) , U 9 37 ( h is t io c y ti c lymphoma), U87 (glioblastoma), and K562 (chronic myelogenous leukemia) were purchased from ATCC. MCF7 cells were obtained from Dr. Kenneth Cowan (NCI) and maintained in Dulbecco’s modified Eagle’s medium containing 10% bovine calf serum and 10 mM Hepes. Tumor tissue was obtained from patients at the time of surgery and snap frozen at 0707C. Informed consent was obtained prior to surgery for the collection of tissue.
Ewing’s sarcoma family of tumors (ESFTs) consists of the ES of bone, extraosseous Ewing’s sarcoma (ES), and peripheral neuroectodermal tumor (PNET) and is characterized by the t(11; 22) (q24; q12) translocation (1–3). ES and PNET are thought to be of neuroectodermal origin because they express neuroectoderm-associated antigens (such as ganglioside GD-2, N-CAM, HNK-1 antigen (4,5)) and because ES cells undergo neuronal differentiation when treated with cyclic adenosine monophosphate or phorbol myristic acid (6). Further, these tumors typically express high levels of choline acetyltransferase and low or undetectable levels of adrenergic enzymes, a characteristic of parasympathetic neurons (7,8).
RNA isolation and RT–PCR. Total RNA was prepared from cultured cells growing in log phase, or from tumor tissue, using RNazol B according to the manufacturer’s recommendations (Tel-Test, Inc.,
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To whom correspondence should be addressed at Clinical Pharmacology Branch, Building 10, Room 13N240, 10 Center Dr. MSC 1928, Bethesda, MD 20892-1928. Fax: (301) 402-0575. Email: [email protected]. 121
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FIG. 1. Expression of PAX3 in various cell lines. (A) 1 mg of total RNA from cells growing in log phase was reverse transcribed and amplified by PCR using PAX3- and b-actin-specific primers. (B) 20 mg of total protein from ESFT cell lines and HeLa and protein produced by in vitro translation of a Pax3 cDNA were analyzed by Western blotting. (C) RDES cells and MCF7 cells were grown on coverslips and stained for PAX3 protein by immunofluorescence. The DNA intercalating dye Hoechst 33258 was used to identify cell nuclei.
Friendswood, TX). One microgram of total RNA was reverse transcribed using random hexamers (Gene Amp RNA PCR kit, Perkin–Elmer Cetus, Foster City, CA). PCR was performed with the upstream primer PP1, 5*-acctcagtcagatgaaggctccgat-3* (exon 5, PAX3), and the downstream primer PP2, 5*-tgaggtctgtggacggtgctactg-3* (exon 7, PAX3). Amplifica-
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tion was performed for 35 cycles at 957C for 1 min, 657C for 1 min, and 727C for 3 min. Twenty percent of the reaction mixture was analyzed on a 2% agarose gel and transferred to Nytran Maximum Strength membrane (Schleicher & Schuell, Keene, NH). Membranes were probed with the end-labeled oligonucleotide PP3, 5*-ACCTCAGTCAGATGAAG-
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GCTCCGAT-3*, which corresponds to a region in exon 5 of PAX3. In order to control for the quality of the RNA preparation and transcription, RT–PCR was performed in parallel with b-actin primers. Experiments were done twice and negative controls were included in every PCR. Western blot analysis. Cell lines were rinsed in phosphate-buffered saline (PBS) and lysed in RIPA buffer (10 mM Tris–HCl, pH 7.2, 0.5% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 1 mM EDTA, 1 mM sodium orthovanadate) containing protease inhibitors (aprotinin 20 mg/ml, leupeptin 20 mg/ml, 1 mM PMSF). Lysates were centrifuged at 14,000g for 20 min at 47C. The protein concentration was measured using the BCA protein assay kit (Pierce, Rockford, IL). Twenty micrograms of total protein was analyzed on an 8% SDS–PAGE minigel as previously described (13). We used a PAX3 rabbit antiserum (14) as the primary antibody. As a posi-
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tive control murine Pax3 was in vitro translated from its full-length cDNA (14) using a rabbit reticulocyte lysate system (Promega, Madison, WI), and an aliquot of this preparation was included in Western blots. The murine Pax3 protein is over 98% homologous with human PAX3 and is recognized by the antibody we used (14). The experiment was performed twice and showed similar results. Immunofluorescence. RDES or MCF7 cells were grown on coverslips. PAX3 was visualized by immunofluorescence as previously described (15) using PAX3 polyclonal rabbit antibody (14). Hoechst 33258 (Molecular Probes, Eugene, OR) was used to visualize cell nuclei. RESULTS We first analyzed various cell lines for PAX3 expression using RT–PCR with PAX3-specific primers
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FIG. 2. Expression of PAX3 in ESFT patient material. RNA was prepared from frozen samples of tumor tissue from five patients with ES and six patients with PNET and used for RT–PCR with PAX3- and b-actin-specific primers. The specificity of the PAX3 PCR product was confirmed by hybridization with a radiolabeled probe.
(Fig. 1A). The specificity of the PCR product was confirmed by hybridization with a specific radiolabeled oligonucleotide probe (data not shown). In order to exclude the amplification of genomic DNA, primers were chosen from exons 5 and 7. PCR amplification in the absence of reverse transcriptase did not produce any detectable signal (data not shown). Several ESFT cell lines were found to express PAX3, including TC32, JR0614, and CHP100. PAX3 was also expressed in cell lines derived from a glioblastoma (U87) and a medulloblastoma (DAOY). A second medulloblastoma (D283) and a neuroblastoma cell line (IMR32) were negative, as were all cell lines derived from hematogenous (Jurkat, K562), lymphomatous (U937), and carcinomatous (Hela, PC3M) tissue (Fig. 1A). Next, we assessed PAX3 protein expression in the ESFT cell lines by Western blotting (Fig. 1B). RDES (ES), TC32 (PNET), and JR0614 (ES) gave a strong signal, while CHP100 expressed low but detectable amounts of PAX3 protein. As a negative control, HeLa cells did not express PAX3 by Western blot analysis. In vitro translated murine Pax3 was used as a positive control (see lane 1 in Fig. 1B). Immunofluorescence analysis of PAX3 expression in RDES cells (Fig. 1C) demonstrated the expected intranuclear localization of PAX3 protein. MCF7 cells served as a negative control. RNA was also prepared from ESFT tumor specimens and used for RT–PCR with PAX3-specific primers. Four of the five tumor specimens (80%) from patients with ES and three of the six tumor specimens (50%) from patients with PNET were found to express PAX3 mRNA (Fig. 2). The specific-
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ity of the PCR product was confirmed by hybridization with a radiolabeled probe. DISCUSSION ES and PNET are thought to be derived from cells of the neural crest (16). PAX3 is expressed in the neural crest during embryogenesis (17,18), as well as in in vitro models of neural crest differentiation (19,20). Since expression of the renal developmental genes PAX2 and PAX8 had been described in Wilms tumor (21–23), we hypothesized that PAX3 could be expressed in ES and PNET. We screened several ES and PNET cell lines and tumor specimens for expression of this paired box gene. We found PAX3 to be expressed in 4/4 ESFT cell lines and 7/11 ESFT tumor specimens. While PAX3 is expressed in a variety of tissues during early embryonal development, including mesencephalon, rhombencephalon, cerebellum, neural tube, neural crest, dermomyotome, and somites (24), expression is very limited in the adult organism (25). For this reason, expression of PAX3 in neuroectodermal tumors is noteworthy. It is impossible to compare the gene expression in ES cells to a normal cell of origin because such a cell has not yet been described (16). PAX genes have been considered protooncogenes because of the expression of gene family members in various tumors and because of certain functional properties of PAX genes. Specifically, high levels of PAX2 and PAX8 have been found in Wilms tumor and renal cell carcinoma (21–23,26), PAX5 was reported in the majority of cases analyzed in medul-
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loblastoma (27) and astrocytoma (28), while PAX8 was expressed in thyroid carcinoma (26,29). Aberrant PAX3 expression has also been implicated in the etiology of alveolar rhabdomyosarcoma, where a characteristic t(2; 13) chromosomal translocation leads to the formation of a hybrid protein that combines the DNA binding domains of PAX3 with the C-terminal activation domain of FKHR, a member of the forkhead family of transcription factors (30,31). Less frequently, the variant t(1; 13) leads to a PAX7/ FKHR fusion in alveolar rhabdomyosarcoma (32). Besides, PAX genes possess functional features that relate to oncogenesis. Data on PAX2 suggest a role of this gene in proliferation of kidney cells (33). The gene is downregulated shortly before or concurrent with terminal differentiation and is found reactivated in regenerating proximal tubule cells. PAX5 was shown to mediate loss of p53 function through transcriptional repression (34). Transfected Pax3 inhibits differentiation in cultured myoblasts (35). And finally, PAX genes were found to transform fibroblasts both in cell culture and in nude mice (36). Although our data suggest that PAX3 expression may be common in ESFT, we observed variability in the level of PAX3 expressed in the different ESFT cell lines, as well as lack of PAX3 expression in several ESFT patient samples. In addition, PAX3 expression is not restricted to this family of tumor, as we were able to demonstrate PAX3 expression in one of two medulloblastoma cell lines examined, as well as in the only glioblastoma cell line studied. Interestingly, however, no PAX3 expression was observed in several carcinoma-, leukemia-, and lymphoma-derived cell lines. At this point the functional significance of PAX3 expression in ESFT cells is not clear. PAX3 could be a lineage marker associated with certain cell types. But given the role PAX3 plays in development, and PAX family genes play in other forms of cancer (27,33,37,38), PAX3 expression may well be involved in oncogenesis. We believe that our findings warrant further study of the expression and function of PAX3 in ESFTs. ACKNOWLEDGMENTS We gratefully thank Dr. David Shapiro (St. Jude Research Hospital, Memphis, TN) for PAX3 antiserum and Pax3 cDNA. This project was supported by the Cooperative Human Tissue Network, which is funded by the National Cancer Institute.
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