The role of cytidine deaminase and GATA1 mutations in the increased cytosine arabinoside sensitivity of Down syndrome myeloblasts and leukemia cell lines.
Myeloblasts from Down syndrome (DS) children with acute myeloid leukemia (AML) are significantly more sensitive in vitro to 1-beta-D-arabinofuranosylcytosine (ara-C) and generate higher 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (ara-CTP) than non-DS AML myeloblasts. Semiquantitative reverse transcription-PCR analyses demonstrated that transcripts for cytidine deaminase (CDA) were 2.7-fold lower in DS than for non-DS myeloblasts. In contrast, transcripts of cystathionine-beta-synthase and deoxycytidine kinase were a median 12.5- and 2.6-fold higher in DS compared with non-DS myeloblasts. The ratio of deoxycytidine kinase/CDA transcripts significantly correlated with ara-C sensitivities and ara-CTP generation. In clinically relevant AML cell line models, high cystathionine-beta-synthase transcripts in DS CMK cells were accompanied by 10-fold greater ara-C sensitivity and 2.4-fold higher levels of ara-CTP compared with non-DS CMS cells. Overexpression of CDA in non-DS THP-1 cells was associated with a 100-fold decreased ara-C sensitivity and 40-fold decreased ara-CTP generation. THP-1 cells secreted CDA into the incubation media and converted extracellular ara-C completely to 1-beta-D-arabinofuranosyluracil within 30 min. Rapid amplification of 5'-cDNA ends (5'-RACE) and reverse transcription-PCR assays identified short- (sf) and long-form (lf) CDA transcripts in THP-1 cells with different 5' untranslated regions and translational start sites; however, only the latter resulted in the active CDA. Although 5' flanking sequences for both CDA transcripts exhibited promoter activity in reporter gene assays, activity for the CDAlf was low. The presence of several GATA1 binding sites in the CDAsf promoter and the uniform detection of GATA1 mutations in DS megakaryocytic leukemia suggested the potential role of GATA1 in regulating CDA transcription and the CDAsf promoter acting as an enhancer. Transfection of GATA1 into Drosophila Mel-2 cells stimulated the CDAlf promoter in a dose-dependent fashion. Additional identification of the mechanisms of differential expression of genes encoding enzymes involved in ara-C metabolism between DS and non-DS myeloblasts may lead to improvements in AML therapy.
Ge
Yubin
Y
Experimental and Clinical Therapeutics Program, Barbara Ann Karmanos Cancer Institute, Division of Pediatric Hematology/Oncology, Children's Hospital of Michigan, Detroit, Michigan, USA.
Jensen
Tanya L
TL
Stout
Mark L
ML
Flatley
Robin M
RM
Grohar
Patrick J
PJ
Ravindranath
Yaddanapudi
Y
Matherly
Larry H
LH
Taub
Jeffrey W
JW
D001483:Base Sequence; D016923:Cell Death / Q000187:drug effects; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D003561:Cytarabine / Q000633:toxicity*; D043525:Cytosine Deaminase / Q000235:genetics* / Q000378:metabolism; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D004314:Down Syndrome / Q000235:genetics / Q000473:pathology*; D050981:Erythroid-Specific DNA-Binding Factors; D005091:Exons / Q000235:genetics; D050982:GATA1 Transcription Factor; D006801:Humans; D007438:Introns / Q000235:genetics; D007938:Leukemia / Q000235:genetics / Q000473:pathology*; D008969:Molecular Sequence Data; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic
D017931:DNA Primers; D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D050982:GATA1 Transcription Factor; C494688:GATA1 protein, human; D014157:Transcription Factors; D003561:Cytarabine; D043525:Cytosine Deaminase