Cloning and characterization of a novel cytokine-inducible protein(p29)

Seiji Fukuda, Ding W. Wu, Louis Pelus

Research output: Contribution to journalArticle

Abstract

Hematopoietic cytokines provide essential stimuli for cell proliferation, differentiation and survival or death. We identified a novel cytokine inducible protein in cell lysates from the Epo-dependent human leukemia cell line, UT7/Epo, using 2-D gel analysis. Compared to Epo-deprived cells, a unique 29kDa spot was consistently observed in lysates from Epo-stimulated cells. After proteolysis and HPLC separation, two peptide sequences were obtained by MALDI-MS that matched a single unknown cDNA in dbEST. The entire ORF was constructed from dbEST consisting of 210 amino acids with a predicted MW of 24kDa. Although P29 lacks known motifs, the N-terminal 45 amino acids show 50% homology with human heterogenous nuclear ribonucleoprotein U (hnRNP) which is associated with messenger RNA processing. P29 is a hydrophilic protein, which is localized in the nucleus as revealed by P29-GFP fusion protein expression in HEK293 cells. P29 has nine potential serine threonine phosphorylation sites and three potential N-glycosylation sites. The gene has at least 9 exons, locates to the long arm of human chromosome 12 and is highly evolutionary conserved from zebrafish to human. It is expressed in most human tissues, indicating that its expression is not unique to Epo. P29 mRNA is high in fetal liver, adult marrow and cardiac muscle, but barely detectable in peripheral blood. Fetal tissues express significantly higher P29 mRNA than adult tissues, and P29 mRNA is several fold higher in cancer cells, particularly leukemia and lymphoma. We have confirmed the expression of P29 mRNA in UT7/Epo cells upon Epo addition and in Mo7e cells upon addition of GMCSF. Furthermore, removal of Epo from UT7/Epo cells results in reduction of P29 mRNA, cell cycle arrest at GJGt and induction of apoptosis. Re-addition of Epo stimulates reexpression of P29 mRNA concomitant with cell cycle progression, suggesting that P29 expression is cell cycle related. P29 overexpression and antisense constructs in HEK293 cells confirm a role for P29 in cell cycle. Synchronization studies in mouse 3T3 cells demonstrated that P29 mRNA remains low in GJGt, but is dramatically up regulated upon entry into S-Phase. In HL-60 cells FACS sorted with respect to cell cycle, high P29 mRNA expression in S and G /M phase cells was observed. We also observed up regulation of P29 mRNA in cord blood CD34+ cells following stimulation by the cytokines SCF, Tpo and Flt3 ligand. These results identify P29 as a new protein that plays a role in proliferation and/or survival of normal hematopoietic and cancer cells.

Original languageEnglish
JournalBlood
Volume96
Issue number11 PART II
StatePublished - 2000

Fingerprint

Cloning
Organism Cloning
Cytokines
Messenger RNA
Cells
Proteins
Cell Cycle
HEK293 Cells
Tissue
Leukemia
Blood
Proteolysis
Glycosylation
Amino Acids
Chromosomes, Human, Pair 12
3T3 Cells
Phosphorylation
Ribonucleoproteins
HL-60 Cells
Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry

ASJC Scopus subject areas

  • Hematology

Cite this

Fukuda, S., Wu, D. W., & Pelus, L. (2000). Cloning and characterization of a novel cytokine-inducible protein(p29). Blood, 96(11 PART II).

Cloning and characterization of a novel cytokine-inducible protein(p29). / Fukuda, Seiji; Wu, Ding W.; Pelus, Louis.

In: Blood, Vol. 96, No. 11 PART II, 2000.

Research output: Contribution to journalArticle

Fukuda, S, Wu, DW & Pelus, L 2000, 'Cloning and characterization of a novel cytokine-inducible protein(p29)', Blood, vol. 96, no. 11 PART II.
Fukuda, Seiji ; Wu, Ding W. ; Pelus, Louis. / Cloning and characterization of a novel cytokine-inducible protein(p29). In: Blood. 2000 ; Vol. 96, No. 11 PART II.
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abstract = "Hematopoietic cytokines provide essential stimuli for cell proliferation, differentiation and survival or death. We identified a novel cytokine inducible protein in cell lysates from the Epo-dependent human leukemia cell line, UT7/Epo, using 2-D gel analysis. Compared to Epo-deprived cells, a unique 29kDa spot was consistently observed in lysates from Epo-stimulated cells. After proteolysis and HPLC separation, two peptide sequences were obtained by MALDI-MS that matched a single unknown cDNA in dbEST. The entire ORF was constructed from dbEST consisting of 210 amino acids with a predicted MW of 24kDa. Although P29 lacks known motifs, the N-terminal 45 amino acids show 50{\%} homology with human heterogenous nuclear ribonucleoprotein U (hnRNP) which is associated with messenger RNA processing. P29 is a hydrophilic protein, which is localized in the nucleus as revealed by P29-GFP fusion protein expression in HEK293 cells. P29 has nine potential serine threonine phosphorylation sites and three potential N-glycosylation sites. The gene has at least 9 exons, locates to the long arm of human chromosome 12 and is highly evolutionary conserved from zebrafish to human. It is expressed in most human tissues, indicating that its expression is not unique to Epo. P29 mRNA is high in fetal liver, adult marrow and cardiac muscle, but barely detectable in peripheral blood. Fetal tissues express significantly higher P29 mRNA than adult tissues, and P29 mRNA is several fold higher in cancer cells, particularly leukemia and lymphoma. We have confirmed the expression of P29 mRNA in UT7/Epo cells upon Epo addition and in Mo7e cells upon addition of GMCSF. Furthermore, removal of Epo from UT7/Epo cells results in reduction of P29 mRNA, cell cycle arrest at GJGt and induction of apoptosis. Re-addition of Epo stimulates reexpression of P29 mRNA concomitant with cell cycle progression, suggesting that P29 expression is cell cycle related. P29 overexpression and antisense constructs in HEK293 cells confirm a role for P29 in cell cycle. Synchronization studies in mouse 3T3 cells demonstrated that P29 mRNA remains low in GJGt, but is dramatically up regulated upon entry into S-Phase. In HL-60 cells FACS sorted with respect to cell cycle, high P29 mRNA expression in S and G /M phase cells was observed. We also observed up regulation of P29 mRNA in cord blood CD34+ cells following stimulation by the cytokines SCF, Tpo and Flt3 ligand. These results identify P29 as a new protein that plays a role in proliferation and/or survival of normal hematopoietic and cancer cells.",
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N2 - Hematopoietic cytokines provide essential stimuli for cell proliferation, differentiation and survival or death. We identified a novel cytokine inducible protein in cell lysates from the Epo-dependent human leukemia cell line, UT7/Epo, using 2-D gel analysis. Compared to Epo-deprived cells, a unique 29kDa spot was consistently observed in lysates from Epo-stimulated cells. After proteolysis and HPLC separation, two peptide sequences were obtained by MALDI-MS that matched a single unknown cDNA in dbEST. The entire ORF was constructed from dbEST consisting of 210 amino acids with a predicted MW of 24kDa. Although P29 lacks known motifs, the N-terminal 45 amino acids show 50% homology with human heterogenous nuclear ribonucleoprotein U (hnRNP) which is associated with messenger RNA processing. P29 is a hydrophilic protein, which is localized in the nucleus as revealed by P29-GFP fusion protein expression in HEK293 cells. P29 has nine potential serine threonine phosphorylation sites and three potential N-glycosylation sites. The gene has at least 9 exons, locates to the long arm of human chromosome 12 and is highly evolutionary conserved from zebrafish to human. It is expressed in most human tissues, indicating that its expression is not unique to Epo. P29 mRNA is high in fetal liver, adult marrow and cardiac muscle, but barely detectable in peripheral blood. Fetal tissues express significantly higher P29 mRNA than adult tissues, and P29 mRNA is several fold higher in cancer cells, particularly leukemia and lymphoma. We have confirmed the expression of P29 mRNA in UT7/Epo cells upon Epo addition and in Mo7e cells upon addition of GMCSF. Furthermore, removal of Epo from UT7/Epo cells results in reduction of P29 mRNA, cell cycle arrest at GJGt and induction of apoptosis. Re-addition of Epo stimulates reexpression of P29 mRNA concomitant with cell cycle progression, suggesting that P29 expression is cell cycle related. P29 overexpression and antisense constructs in HEK293 cells confirm a role for P29 in cell cycle. Synchronization studies in mouse 3T3 cells demonstrated that P29 mRNA remains low in GJGt, but is dramatically up regulated upon entry into S-Phase. In HL-60 cells FACS sorted with respect to cell cycle, high P29 mRNA expression in S and G /M phase cells was observed. We also observed up regulation of P29 mRNA in cord blood CD34+ cells following stimulation by the cytokines SCF, Tpo and Flt3 ligand. These results identify P29 as a new protein that plays a role in proliferation and/or survival of normal hematopoietic and cancer cells.

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