Effect of 1-beta-D-arabinofuranosylcytosine on apoptosis and differentiation in human monocytic leukemia cells (U937) expressing a c-Jun dominant-negative mutant protein (TAM67).
The proto-oncogene c-jun encodes a component of the AP-1 transcription-activating complex and has been implicated in the regulation of diverse cellular processes, including cell proliferation, differentiation, transformation, and most recently, apoptosis. We have used a U937 monocytic leukemia cell line stably expressing a c-jun dominant-negative, transactivation-domain deletion mutant (TAM67) to assess the role of c-jun in apoptotic events induced by exposure to the antimetabolite 1-beta-D-arabinofuranosylcytosine (ara-C). Mutant cells produce a truncated M(r) 29,000 protein that interferes with the function of normal c-Jun (and c-Fos) proteins through a quenching mechanism. Parental U937, cells expressing TAM67, and cells carrying only the empty vector (pMM) were exposed to ara-C for 6 h, and apoptosis was monitored by cell morphology as well as qualitative and quantitative assays of DNA damage. No differences in apoptosis could be detected between the three cell lines at any of the ara-C concentrations evaluated. In addition, ara-C concentrations > or = 1.0 x 10(-6) M were equally inhibitory to the clonogenic growth of U937 and TAM67-expressing cells. In contrast, lower concentrations of ara-C (i.e., < 5.0 x 10(-7) M) were significantly less inhibitory to mutant U937 cell colony formation than to their parental counterparts. The reduced sensitivity of TAM67-expressing cells to low concentrations of ara-C could not be attributed to biochemical or cytokinetic factors, since the two cell lines were indistinguishable with respect to 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (ara-CTP) formation, ara-CTP:dCTP ratios, and S-phase fraction. However, a significantly lower percentage of TAM67-expressing cells exposed to submicromolar concentrations of ara-C exhibited features associated with a differentiated monocytoid phenotype (i.e., increased plastic adherence and CD11b expression) compared to their parental counterparts. Lower concentrations of ara-C were also significantly less effective in decreasing the percentage of S-phase cells and in down-regulating c-myc mRNA levels in the mutant line, events associated with induction of leukemic cell differentiation. Finally, ara-C-induced up-regulation of c-jun message and protein was markedly attenuated in TAM67-expressing cells, findings consistent with a c-jun dominant-negative model. Collectively, these findings suggest that dysregulation of c-jun in U937 cells antagonizes low-dose ara-C-mediated cellular maturation but does not prevent higher concentration of this agent from triggering apoptosis. They also raise the possibility that separate aspects of the antiproliferative actions of ara-C may be differentially regulated by c-jun.
Grant
S
S
Department of Medicine, Medical College of Virginia, Richmond 23298, USA.
Freemerman
A J
AJ
Birrer
M J
MJ
Martin
H A
HA
Turner
A J
AJ
Szabo
E
E
Chelliah
J
J
Jarvis
W D
WD
D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D002453:Cell Cycle / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D003561:Cytarabine / Q000494:pharmacology*; D003603:Cytotoxins / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D015473:Leukemia, Promyelocytic, Acute / Q000473:pathology*; D009154:Mutation / Q000502:physiology; D000090063:Proto-Oncogene Mas; D016755:Proto-Oncogene Proteins c-jun / Q000235:genetics*; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D014407:Tumor Cells, Cultured / Q000166:cytology / Q000187:drug effects
D000964:Antimetabolites, Antineoplastic; D003603:Cytotoxins; C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D016755:Proto-Oncogene Proteins c-jun; D016271:Proto-Oncogene Proteins c-myc; D012333:RNA, Messenger; D003561:Cytarabine