Activation of the early growth response 1 gene and nuclear pp90rsk in human myeloid leukemia cells by 1-(beta-D-arabinofuranosyl)cytosine.
The early growth response 1 (EGR-1) gene is induced by mitogens, differentiating stimuli, and certain genotoxic agents in diverse cell types. The present work has examined the effects of 1-(beta-D-arabinofuranosyl)cytosine (ara-C), an antileukemia agent that misincorporates into DNA, on EGR-1 expression. Treatment of HL-525 myeloid leukemia cells with ara-C was associated with transient increases in EGR-1 mRNA levels. Nuclear run-on assays showed that this effect is related at least in part to activation of EGR-1 gene transcription. Sequences responsive to ara-C-induced signals were determined by deletion analysis of the EGR-1 promoter. The results demonstrate that ara-C inducibility of the EGR-1 gene is conferred by a region containing six serum response or CC(A/T)6GG (CArG) motifs. Further analysis demonstrated that the first two distal or 5'-most CArG elements are functional in the ara-C response. An oligomer corresponding to the first CArG element also conferred ara-C inducibility of the minimal thymdine kinase gene promoter, while no inducibility was detectable using a similar oligomer containing a mutated CArG box. Other work has demonstrated that the nuclear serum response factor (SRF) interacts with the CArG box in the EGR-1 promoter and that the serine/threonine pp90rsk protein kinase phosphorylates SRF in vitro at sites phosphorylated in vivo. The present studies demonstrate that ara-C has little if any effect on cytosolic pp90rsk as determined by immunoblotting to assess electrophoretic mobility and by immune-complex kinase assays using S6 peptide as substrate.(ABSTRACT TRUNCATED AT 250 WORDS)
Kharbanda
S
S
Laboratory of Clinical Pharmacology, Dana-Farber Cancer Institute, Boston, Massachusetts.
Saleem
A
A
Rubin
E
E
Sukhatme
V
V
Blenis
J
J
Kufe
D
D
D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000235:genetics; D003561:Cytarabine / Q000494:pharmacology*; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D051766:Early Growth Response Protein 1; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D006801:Humans; D017874:Immediate-Early Proteins; D007951:Leukemia, Myeloid / Q000235:genetics*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis / Q000235:genetics*; D012313:RNA / Q000302:isolation & purification; D012333:RNA, Messenger / Q000032:analysis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D038601:Ribosomal Protein S6; D019893:Ribosomal Protein S6 Kinases; D012269:Ribosomal Proteins / Q000378:metabolism; D017384:Sequence Deletion; D026362:Serum Response Factor; D013347:Subcellular Fractions; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; D017874:Immediate-Early Proteins; D009687:Nuclear Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D038601:Ribosomal Protein S6; D012269:Ribosomal Proteins; D026362:Serum Response Factor; D014157:Transcription Factors; D003561:Cytarabine; D012313:RNA; D015500:Chloramphenicol O-Acetyltransferase; D017346:Protein Serine-Threonine Kinases; D019893:Ribosomal Protein S6 Kinases