Pescadillo, a Novel Cell Cycle Regulatory Protein Abnormally Expressed in Malignant Cells

Using a culture model of glial tumorigenesis, we identified a novel gene that was up-regulated in malignant mouse astrocytes following the loss of p53. The gene represents the murine homologue of pescadillo, an uncharacterized gene that is essential for embryonic development in zebrafish. Pescadillo is a strongly conserved gene containing unique structural motifs such as a BRCA1 C-terminal domain, clusters of acidic amino acids and consensus motifs for post-translational modification by SUMO-1. Pescadillo displayed a distinct spatial and temporal pattern of gene expression during brain development, being detected in neural progenitor cells and postmitotic neurons. Although it is not expressed in differentiated astrocytes in vivo, the pescadillo protein is dramatically elevated in malignant human astrocytomas. Yeast strains harboring temperature-sensitive mutations in the pescadillo gene were arrested in either G1 or G2 when grown in nonpermissive conditions, demonstrating that pescadillo is an essential gene in yeast and is required for cell cycle progression. Consistent with the latter finding, DNA synthesis was only observed in mammalian cells expressing the pescadillo protein. These results suggest that pescadillo plays a crucial role in cell proliferation and may be necessary for oncogenic transformation and tumor progression. Using a culture model of glial tumorigenesis, we identified a novel gene that was up-regulated in malignant mouse astrocytes following the loss of p53. The gene represents the murine homologue of pescadillo, an uncharacterized gene that is essential for embryonic development in zebrafish. Pescadillo is a strongly conserved gene containing unique structural motifs such as a BRCA1 C-terminal domain, clusters of acidic amino acids and consensus motifs for post-translational modification by SUMO-1. Pescadillo displayed a distinct spatial and temporal pattern of gene expression during brain development, being detected in neural progenitor cells and postmitotic neurons. Although it is not expressed in differentiated astrocytes in vivo, the pescadillo protein is dramatically elevated in malignant human astrocytomas. Yeast strains harboring temperature-sensitive mutations in the pescadillo gene were arrested in either G1 or G2 when grown in nonpermissive conditions, demonstrating that pescadillo is an essential gene in yeast and is required for cell cycle progression. Consistent with the latter finding, DNA synthesis was only observed in mammalian cells expressing the pescadillo protein. These results suggest that pescadillo plays a crucial role in cell proliferation and may be necessary for oncogenic transformation and tumor progression. green fluorescent protein polyacrylamide gel electrophoresis bromodeoxyuridine polymerase chain reaction fluorescence-activated cell sorting BRCA1 C terminus Astrocyte-derived neoplasms represent the most common type of primary central nervous system tumor. Recent studies have been directed toward defining the genes and gene products responsible for glial tumorigenesis and progression. Mutation or loss of the tumor suppressor gene p53 is thought to be an important event in the early neoplastic transformation of astrocytes (1Nagane M. Huang H.J. Cavenee W.K. Curr. Opin. Oncol. 1997; 9: 215-222Crossref PubMed Scopus (60) Google Scholar, 2Rasheed B.K. Wiltshire R.N. Bigner S.H. Bigner D.D. Curr. Opin. Oncol. 1999; 11: 162-167Crossref PubMed Scopus (195) Google Scholar). Studies of human glial tumors have demonstrated that loss of the p53 gene is a frequent event in all grades of astrocytoma. A subpopulation of cells will then proceed to either mutate or delete the remaining copy of the p53 gene, leading to uncontrolled cellular proliferation. Of interest are studies suggesting that some high grade astrocytic malignancies result from clonal expansion of cells that have lost or mutated their p53 (3Sidransky D. Mikkelsen T. Schwechheimer K. Rosenblum M.L. Cavenee W. Vogelstein B. Nature. 1992; 355: 846-847Crossref PubMed Scopus (633) Google Scholar). The genes that may be adversely regulated following p53 alterations and that might contribute to enhanced growth and survival are just now being identified in different biological contexts but not yet in malignant astrocytes (4Polyak K. Xia Y. Zweier J.L. Kinzler K.W. Vogelstein B. Nature. 1997; 389: 300-305Crossref PubMed Scopus (2246) Google Scholar, 5Yu J. Zhang L. Hwang P.M. Rago C. Kinzler K.W. Vogelstein B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14517-14522Crossref PubMed Scopus (416) Google Scholar, 6Zhao R. Gish K. Murphy M. Yin Y. Notterman D. Hoffman W.H. Tom E. Mack D.H. Levine A.J. Genes Dev. 2000; 14: 981-993Crossref PubMed Scopus (277) Google Scholar). We have previously established, using cultured astrocytes from p53-deficient mice, an in vitro model of malignant transformation recapitulating glial tumorigenesis (7Yahanda A.M. Bruner J.M. Donehower L.A. Morrison R.S. Mol. Cell. Biol. 1995; 15: 4249-4259Crossref PubMed Scopus (91) Google Scholar). Early passage p53−/− astrocytes achieved higher saturation densities than p53+/− and p53+/+ cells but did not exhibit evidence of neoplastic transformation. With continued passaging, p53−/− astrocytes exhibit a multistep progression to a transformed phenotype displaying significant aneuploidy and acquiring the ability to form large, well vascularized tumors in nude mice. In marked contrast, p53+/+ astrocytes fail to show a transformed phenotype and senesce after 7–10 passages. Thus, loss of wild type p53 function promotes genomic instability, accelerated growth, and malignant transformation in astrocytes. Using this model system in conjunction with cDNA microarray technology, we sought to examine the relationship between the progressive phenotypic changes in astrocytes and alterations in gene expression. Glass-based high density microarray hybridization is an efficient way to establish a detailed expression profile of up to 10,000 genes simultaneously from a single tissue or cell type and has been successfully used to analyze tumor- or tissue-specific patterns of gene expression (8Epstein C.B. Butow R.A. Curr. Opin. Biotechnol. 2000; 11: 36-41Crossref PubMed Scopus (114) Google Scholar, 9Khan J. Bittner M.L. Chen Y. Meltzer P.S. Trent J.M. Biochim. Biophys. Acta. 1999; 1423: M17-M28PubMed Google Scholar). We have utilized cDNA arrays to identify genes that are differentially expressed between malignant late passage p53−/− astrocytes and nontransformed early passage p53+/+ and p53+/−astrocytes. 1G. Foltz, J. Schuster, Y. Kinoshita, L. Hood, R. S. Morrison, and P. S. Nelson, manuscript in preparation One expressed sequence tag that was up-regulated in malignant p53−/−astrocytes represented the murine homologue of a novel zebrafish gene known as pescadillo (10Allende M.L. Amsterdam A. Becker T. Kawakami K. Gaiano N. Hopkins N. Genes Dev. 1996; 10: 3141-3155Crossref PubMed Scopus (154) Google Scholar). The pescadillo gene was initially identified through an embryonic mutation in zebrafish, which resulted in animals bearing small eyes, impaired brain growth, and aberrant development of the liver and gut. Pescadillo is widely and highly expressed during the first 3 days of zebrafish development but was not observed in any adult tissues except for the ovary, suggesting that its expression is principally limited to developing tissues. In the present study, we present an analysis of the pescadillo protein and provide evidence that pescadillo is a nuclear cell cycle regulatory protein that is abnormally expressed in malignant astrocytes and other transformed cell types. COS-7, HeLa, and SW480 colon carcinoma cells were obtained from the American Type Tissue Culture Collection. SNB-19 human glioblastoma cells (11Welch W.C. Morrison R.S. Gross J.L. Gollin S.M. Kitson R.B. Goldfarb R.H. Giuliano K.A. Bradley M.K. Kornblith P.L. In Vitro Cell Dev. Biol. Anim. 1995; 31: 610-616Crossref PubMed Scopus (21) Google Scholar) and p53+/+, p53+/−, and p53−/− astrocytes were prepared and maintained as described previously (7Yahanda A.M. Bruner J.M. Donehower L.A. Morrison R.S. Mol. Cell. Biol. 1995; 15: 4249-4259Crossref PubMed Scopus (91) Google Scholar). Normal human mammary epithelial cells and MCF-7 human breast carcinoma cells were generously provided by Dr. Karen Swisshelm (Department of Pathology, University of Washington). A primary culture of human glioblastoma cells was established from a primary glioblastoma resected at the University of Washington by previously described methods (12Chicoine M.R. Silbergeld D.L. Cancer. 1995; 75: 2904-2909Crossref PubMed Scopus (57) Google Scholar). The procedure for obtaining and culturing human tumor tissue received human subjects approval from the Institutional Review Board Committee of the University of Washington, and informed consent was obtained from the patient prior to surgery. All cells were routinely maintained in Dulbecco's modified Eagle's medium/Ham's F-12 medium, except for HeLa cells, which were maintained in Dulbecco's modified Eagle's medium, with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin at 37 °C in 5% CO2. Expression constructs encoding pescadillo fused to the enhanced green fluorescent protein (GFP)2 or to a Myc epitope were created using a full-length human pescadillo cDNA (derived from a Soares human fetal brain cDNA library obtained from Research Genetics) inserted into the vector pEGFP-C3 (CLONTECH) (13Hsu Y.T. Wolter K.G. Youle R.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3668-3672Crossref PubMed Scopus (1032) Google Scholar) or a Myc-tagged expression vector driven by the cytomegalovirus promoter (Cs2+Myc tag) A.M. J. Cell Biol. PubMed Scopus Google DNA were using a M. J. R.J. 1995; PubMed Scopus Google Scholar) as we described previously S. S. Y. M. L. Xia Youle R.J. Morrison R.S. J. Cell Biol. 2000; PubMed Scopus Google Scholar). days after cells were with and using or cells were for A was a from the C-terminal amino acids of the human pescadillo protein and used after was obtained from were prepared from tumor and brain tissue as described previously A. Mol. 1995; PubMed Scopus Google Scholar). cells at were in an as described previously Y. S. Morrison R.S. J. 1999; PubMed Google Scholar). glioblastoma and as well as brain tissue the primary tumors were generously provided by Dr. from the of of Washington). tissue is routinely to the of All provided informed their tissue to be used for was as described previously Y. S. Morrison R.S. J. 1999; PubMed Google Scholar) with The were at °C with an an or an epitope culture J.M. Mol. Cell. Biol. PubMed Scopus Google by were with a mouse to for protein The of the protein to pescadillo and the was by using analysis cells were with the and protein were prepared days as described for were with epitope culture with protein at protein were with the for 3 at °C and then by using for at The were with and in electrophoresis The were by by and with an as were cultured in with at for and then with for in a containing 5% serum, bovine in for at were with primary at by with at for at were in and an with were and into of a was in single and the of was in an way for was used at at and Studies at were with for at In was as described previously K. Mol. PubMed Scopus Google Scholar). animals were by and their or were in and a at in and at A cDNA encoding the mouse pescadillo gene was into was prepared from the and using polymerase in the of with polymerase and were used as Tissue were and with the A were in of and to prior to in The were then and with A of the yeast pescadillo homologue was using a gene R.S. E. J. J. 1995; PubMed Scopus Google Scholar). gene was from with containing to the and of The was transformed in yeast was in by were and were in for All were that is an essential The temperature-sensitive mutations were using a procedure and into the at the C. P. Cell. 1996; PubMed Scopus Google Scholar). a DNA from a yeast genomic was into which was then used as a for and were into vector and at the and in at °C were and at either or 37 °C for 3 and to DNA analysis by fluorescence-activated cell sorting P. J. 9: PubMed Scopus Google Scholar) or with and to nuclear and using fluorescent p53−/− astrocytes maintained in culture for or the ability to form in and form large, well vascularized tumors in nude mice, in to p53+/+ astrocytes and early passage p53+/− astrocytes (7Yahanda A.M. Bruner J.M. Donehower L.A. Morrison R.S. Mol. Cell. Biol. 1995; 15: 4249-4259Crossref PubMed Scopus (91) Google Scholar). We to changes in gene expression that were with different using high density cDNA We observed a of genes expression pattern was as a result of proliferation in the of One cDNA in was expressed at higher in late passage p53−/− astrocytes to early passage p53−/− or and the sequence of an uncharacterized gene to as pescadillo (10Allende M.L. Amsterdam A. Becker T. Kawakami K. Gaiano N. Hopkins N. Genes Dev. 1996; 10: 3141-3155Crossref PubMed Scopus (154) Google Scholar). The pescadillo gene was for in malignant astrocytes its expression was to of embryonic development, and it was to be essential for nervous system development in zebrafish. The human pescadillo gene is well conserved a of and and sequence with yeast and zebrafish pescadillo The zebrafish, and yeast pescadillo in from to amino and unique structural motifs a BRCA1 C-terminal domain, acidic amino clusters and in the C and conserved consensus for of M. Cell Biol. 1997; PubMed Scopus Google Scholar) All pescadillo nuclear suggesting that pescadillo may function in the The of the pescadillo protein was by and by expressing a Myc or pescadillo detected by an a C-terminal was observed as highly in the with to be with when which was by with an and The of pescadillo protein was with the pattern obtained after expressing of pescadillo, and nuclear was observed in cell type with the human glioblastoma cells, HeLa cells, human and p53−/− mouse astrocytes not These results the of the as well as the of the of postmitotic primary in culture displayed a different pattern than that observed in the cell the was the not These results that pescadillo is a nuclear protein the may be regulated in a cell The for the human pescadillo protein is of prepared from cells with human and to using an displayed to of and were in prepared from cells with the Myc-tagged The protein was a and was observed in cell not to The to the of the full-length pescadillo protein the Myc in the expression We that the represented a modified form of pescadillo it was only observed in We this in the following The pescadillo protein sequence was for the of post-translational modification We observed the of consensus for the modification by the M. Cell Biol. 1997; PubMed Scopus Google Scholar). The most highly conserved was at The of the was with the of the which has been to the of by J. J. Cell Biol. PubMed Scopus Google Scholar). for the of the were using prepared from cells with and to using an The was with an and only the was detected results were obtained when was with the and the was with the not In to the results obtained from pescadillo the represented the form of pescadillo protein in a human glioblastoma cell cells and in late passage p53−/− mouse astrocytes A at with the of was detected in cell a was not These results the and of the pescadillo and that some the pescadillo protein may be modified by the of SUMO-1. The cellular and the temporal pattern of pescadillo expression were in the mammalian brain to which cell in the brain Using in hybridization the pescadillo was to be widely and highly expressed the developing mouse brain and at embryonic with its expression in neural progenitor cells and developing 3 hybridization was detected with the or in tissue with A not the of hybridization was present in the and developing with hybridization detected in the developing and hybridization was present developing such as the 3 development, the pattern of did not to not In the was detected in not of the but with some The most hybridization was present in such as the in neurons. Pescadillo was present the of the the 3 is the of in the adult or hybridization was present in the adult suggesting that glial cells not The in hybridization studies described that pescadillo is not expressed by differentiated astrocytes in to that pescadillo is abnormally expressed in malignant astrocytes as from cDNA microarray we the expression of the pescadillo protein in and transformed mouse astrocytes in of pescadillo were in primary of p53−/− and and p53+/+ astrocytes not previously p53+/+ astrocytes and early passage p53−/− astrocytes a pattern of growth and fail to form tumors in nude (7Yahanda A.M. Bruner J.M. Donehower L.A. Morrison R.S. Mol. Cell. Biol. 1995; 15: 4249-4259Crossref PubMed Scopus (91) Google Scholar). Pescadillo in primary of p53−/−astrocytes was to the and in the pattern that was observed following with pescadillo in of cell density or of in culture to 3 not In marked contrast, pescadillo was elevated in late passage p53−/− mouse astrocytes and the the in malignant astrocytes. of pescadillo protein expression was observed in MCF-7 human breast carcinoma cells and with human mammary epithelial cells and significant of pescadillo were detected in a of human cell colon carcinoma and carcinoma cells cells, and that pescadillo expression was not elevated in established cell of growth in we pescadillo expression in a primary culture of human glioblastoma cells that was of a resected tumor being The primary glioblastoma culture expressed the nuclear pescadillo that was in the established cell and that pescadillo expression may be elevated in malignant astrocytes in We this by the of pescadillo protein in tissue resected from of malignant brain tumor tissue and the brain tissue the tumor Pescadillo was in brain tissue obtained from neoplastic demonstrated that the pescadillo protein was highly expressed in malignant human glial tumors and pescadillo expression were expression the malignant tumors up to a in pescadillo expression to the in pescadillo expression between may the that tissue is of glial cells and of tumor These that the pescadillo protein is abnormally elevated in malignant human tumors of astrocytic The C has been identified in a of in of DNA and P. K. P. J. 1997; 11: PubMed Scopus Google Scholar, 1997; PubMed Scopus Google Scholar). identified a in the pescadillo we pescadillo was with cell cycle progression. that pescadillo expression was when cells were maintained as for HeLa cells A which was with proliferation as by the relationship between cell density and pescadillo HeLa cells grown to a high density A and were at different at a density of the cells pescadillo and of the cells by after In contrast, when the cells were at a density that the density the cells a in pescadillo and to when a of cells was from the HeLa cell cells that into the pescadillo with These results that is only observed in cells expressing pescadillo suggesting that pescadillo may be required for progression through the cell evidence that pescadillo is necessary for cell cycle progression was obtained by the yeast pescadillo homologue The human pescadillo protein significant with the the and In the yeast homologue protein to the not suggesting that human pescadillo may the function as its yeast of the in a yeast and demonstrated that is an essential into the function of temperature-sensitive of were by and into the a the yeast strains were to Cell cycle progression of the strains was by their DNA by and cell by °C wild type and yeast strains the at 37 °C a G1 and a G2 Cell analysis the results obtained at the nonpermissive that of cells in the G1 of cells a with the at the and a to a in analysis that strains a single mutation in the for for as well as and of the this that pescadillo is necessary for cell cycle progression and that the is essential for pescadillo of the tumor suppressor gene p53 has been in the of human We to changes neoplastic transformation in vitro mouse model of tumorigenesis (7Yahanda A.M. Bruner J.M. Donehower L.A. Morrison R.S. Mol. Cell. Biol. 1995; 15: 4249-4259Crossref PubMed Scopus (91) Google Scholar). this we that the murine homologue of pescadillo was highly expressed in malignant mouse astrocytes. The of this novel protein that pescadillo expression in the mouse brain is to neural progenitor cells and postmitotic but is in malignant mouse astrocytes in vitro following growth in the of that pescadillo expression is elevated in malignant human in that the loss of p53 is necessary but not by to pescadillo expression in and that pescadillo is a nuclear protein necessary for cell cycle progression. results that a novel of embryonic development in zebrafish, pescadillo, is expressed in a of malignant mammalian results that pescadillo protein expression is elevated in malignant murine and malignant human astrocytes in the pescadillo protein is expressed at higher in malignant human brain tumors to the tissue from the patient and brain tissue obtained from neoplastic The tissue a brain tumor is of cells with of tumor cells and cells, suggesting that pescadillo in the tumor expression by malignant astrocytes. is with the pescadillo detected in primary and established cell from human pescadillo expression was observed in neural progenitor cells but not in differentiated it is that glial tumors from pescadillo progenitor glial tumors from differentiated astrocytes with pescadillo expression. of the cell of results that pescadillo is abnormally expressed in malignant human tumors of astrocytic Pescadillo protein was identified in a of malignant human cell D. and R. S. Morrison, and colon carcinoma cell is the significant in pescadillo that was detected in malignant human breast carcinoma cells to human mammary epithelial late passage malignant murine p53−/− astrocytes higher of pescadillo than p53+/+ or nontransformed early passage p53−/− astrocytes. Although the p53 of cell is not the results obtained with nontransformed early passage p53−/−astrocytes suggest that the loss of p53 is not to pescadillo expression. The human pescadillo gene has been to N. S. R. M. R. A. C. J. K. S. A.M. D. J. 1999; PubMed Scopus Google Scholar). are common in human but may to the pescadillo a common of at Y. L. R. M. A. J. 1999; PubMed Scopus (57) Google Scholar). of the human has not been suggesting that of the pescadillo protein in malignant are not the result of gene In the of pescadillo in malignant mouse astrocytes is small to in pescadillo Schuster, Foltz, P. S. Nelson, and R. S. Morrison, changes may not pescadillo gene but that pescadillo or protein of the pescadillo in malignant cells is not to cultured tumor cells as demonstrated by its elevated expression in malignant human glial These that pescadillo may a function that is essential for the malignant The function by pescadillo has not been but into its cellular role be from structural motifs the pescadillo protein The most in pescadillo is a protein identified in the breast and gene BRCA1 P. K. P. J. 1997; 11: PubMed Scopus Google Scholar, 1997; PubMed Scopus Google Scholar). A that has from the that a is that of in DNA P. K. P. J. 1997; 11: PubMed Scopus Google Scholar, 1997; PubMed Scopus Google Scholar). conserved consensus for the of were identified in pescadillo, and modification was demonstrated in the present Although the with modification are is evidence that may be important for protein and of the protein to the nuclear is the modification J. J. Cell Biol. PubMed Scopus Google Scholar). The to with important regulatory such as the of the and tumor T. L. T. T. J. 1996; Google Scholar). of and tumor cell suggesting that the of T. L. T. T. J. 1996; Google Scholar). The protein has been to in a with human and a Chen 1996; PubMed Scopus Google Scholar). a is thought to be in DNA and of These results that the by modified by in protein that cellular essential for cell proliferation and In of this it has been that is essential for in S. and is required for into J. Cell Biol. 1999; PubMed Scopus Google Scholar). The of function observed between expressing a and modified by is with the that pescadillo is in the of a cell cycle The of pescadillo to the of cell growth was by the and of temperature-sensitive pescadillo yeast Yeast expressing pescadillo displayed growth in the G1 or G2 of the cell cycle when to a nonpermissive yeast strains a single mutation in the domain, the of this to pescadillo is not the strains in different of the cell The which in an mutation at amino to the of the mutation the may pescadillo or its The results obtained with the yeast that pescadillo is essential for cell cycle for the between pescadillo expression and progression observed in HeLa The of such a cell cycle gene during for the tissue observed in zebrafish a pescadillo gene (10Allende M.L. Amsterdam A. Becker T. Kawakami K. Gaiano N. Hopkins N. Genes Dev. 1996; 10: 3141-3155Crossref PubMed Scopus (154) Google Scholar). The results of the present that pescadillo, a novel gene expressed principally in developing is expressed in adult human malignant brain Pescadillo is up-regulated in different carcinoma cell suggesting that in pescadillo expression may be a common of pescadillo in nontransformed cell such as cells, cell of ability or growth, and R. S. Morrison, suggesting that pescadillo is not oncogenic in nontransformed cells but may be necessary for the of that progression through the cell In to nontransformed cells, pescadillo did not the or growth of malignant cell which high of the pescadillo Thus, the function of pescadillo in cell cycle progression and of pescadillo function may unique into the of neoplastic transformation and provide a for We for and T. for the The was obtained from the Studies maintained by the University of (Department of

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