Spontaneous down-regulation of Fas-associated phosphatase-1 may contribute to excessive apoptosis in myelodysplastic marrows.
In this study, we examined the role of Fas-signaling in the apoptotic pathway in myelodysplastic syndromes (MDS). Ficoll-separated mononuclear cells from 18 bone marrow aspirate specimens obtained from 17 MDS patients, 4 normal healthy donors, and 3 acute myeloid leukemia patients transformed from MDS (t-AML) were studied for mRNA expression of Fas-L, Fas, and the effectors of their signaling, Caspase 1 and Caspase 3, using reverse transcriptase polymerase chain reaction. Fas-L, Fas, and Caspase 1 were detectable in all of the samples in the three groups. Caspase 3 was detectable both in MDS and t-AML specimens but was negligible in normal cells. The apoptotic index (AI%) determined by in situ end labeling of fragmented DNA in 4-hour cultures of mononuclear cells was significantly higher in MDS cells compared to normal or t-AML cells (mean +/- SEM: 2.3% +/- 0.4% in MDS, n = 10 vs. 0.6% +/- 0.2%, n = 4, P = 0.014 in normal cells, and 0.2% +/- 0.2%, n = 3, P = 0.007 in t-AML cells). Treatment of MDS cells with anti-Fas-L antibody suppressed apoptosis (AI%: 2.1% +/- 0.6% in untreated vs. 1.37% +/- 0.5% in treated, n = 6, P = 0.02), indicating functional participation of Fas-signaling in MDS. Further, it was found that Fas-L, Fas, and Caspase 1 mRNA expression remained unchanged in 4 hours. Caspase 3 expression appeared in normal cells after 4 hours and was present at both 0 and 4 hours in MDS and t-AML cells. In contrast to persistent expression in normal and t-AML cells, cells from the 5 MDS patients studied consistently showed significantly lowered or undetectable expression of a negative regulator of Fas, called Fas-associated phosphatase-1 (Fap-1) after 4 hours. Thus, the high AI% in MDS corresponds to a rapid decline in Fap-1. Furthermore, in tumor necrosis factor alpha (TNF-alpha) treated HL60 promyelocytic cells, a definite periodicity in the expression of different mRNAs was observed with upregulation of TNF-alpha itself at 30 minutes, increased expression of Fas and the appearance of Fas-L after 2 hours, and a decrease in Fap-1 expression after 8 hours. These results suggest that TNF-alpha not only induces the effectors of Fas-signaling but also may downregulate the inhibitor. We conclude that a spontaneous and rapid down-regulation of Fap-1, possibly induced by TNF-alpha, a cytokine shown to be present in excess in MDS marrows, may underlie the increased apoptotic death of hematopoietic cells in these patients. Interference with Fap-1 turnover may provide a new therapeutic modality for MDS.
Mundle
S D
SD
Rush Cancer Institute, Rush-Presbyterian-St. Luke's Medical Center, Chicago, Illinois 60612, USA. smundle@rush.edu
Mativi
B Y
BY
Bagai
K
K
Feldman
G
G
Cheema
P
P
Gautam
U
U
Reza
S
S
Cartlidge
J D
JD
Venugopal
P
P
Shetty
V
V
Gregory
S A
SA
Robin
E
E
Rifkin
S
S
Shah
R
R
Raza
A
A
D017209:Apoptosis / Q000235:genetics; D001853:Bone Marrow / Q000201:enzymology / Q000473:pathology; D002352:Carrier Proteins / Q000378:metabolism*; D015536:Down-Regulation; D004789:Enzyme Activation; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D009190:Myelodysplastic Syndromes / Q000201:enzymology* / Q000235:genetics / Q000473:pathology*; D054645:Protein Phosphatase 1; D054595:Protein Tyrosine Phosphatase, Non-Receptor Type 13; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D019014:fas Receptor / Q000378:metabolism
D002352:Carrier Proteins; D019014:fas Receptor; D054645:Protein Phosphatase 1; C516742:PTPN13 protein, human; D054595:Protein Tyrosine Phosphatase, Non-Receptor Type 13; D017027:Protein Tyrosine Phosphatases