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Title: Spontaneous down-regulation of Fas-associated phosphatase-1 may contribute to excessive apoptosis in myelodysplastic marrows.
Author: Mundle SD, Mativi BY, Bagai K, Feldman G, Cheema P, Gautam U, Reza S, Cartlidge JD, Venugopal P, Shetty V, Gregory SA, Robin E, Rifkin S, Shah R, Raza A.
Journal: Int J Hematol; 1999 Aug; 70(2):83-90. PubMed ID: 10497846.
Abstract:
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.

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