Quantitative analysis of Fas and bcl-2 expression in hematopoietic precursors.
OBJECTIVE
We investigated the expression of bcl-2 and CD95 (Apo1-/Fas) on CD34+ cells obtained from bone marrow (BM), mobilized peripheral blood (MPB), and umbilical cord blood (UCB) samples. The expression of bcl-2 and Fas was then compared with that of other markers usually associated with immaturity; functional tests using the agonistic antibody anti- Fas CH11 were also carried out.


METHODS
The analysis was performed by flow cytometry on purified CD34+ cells in a three (CD95 PE, CD34 APC and CD71 FITC) and in a four (CD38 PE, HLA-DR PerCP, CD34 APC and bcl-2 FITC) fluorescence assay.


RESULTS
The results were expressed as mean fluorescence index (MFI); bcl-2 expression was significantly higher (p < 0.001) in BM (3.73 +/- 0.63) than in MPB (2.47 +/- 0.39) and UCB (2.38 +/- 0.58); Fas was significantly less expressed (p < 0.001) in UCB (1.27 +/- 0.78) than in MBP (3.63 +/- 2.19) and BM (4.56 +/- 1.69). CD34 expression was significantly (p < 0.001) brighter in UCB compared to in MBP and BM, while CD38 and CD71 were significantly (p = 0.005 and p < 0.001, respectively) more expressed in BM than in MPB and UCB. Fas values were directly correlated to CD38; both Fas and bcl-2 were directly related to CD71 and inversely to CD34. Culture assays showed that hematopoietic precursor cells from BM, MPB and UCB had a low susceptibility to undergo Fas-mediated apoptosis.


CONCLUSIONS
In conclusion, bcl-2 and Fas are less expressed in UCB than in MPB and BM; early hematopoietic precursor cells are relatively resistant to CD95-triggered apoptosis; the observed correlation between Fas/bcl-2 and markers of immaturity suggests that they may be determinants of commitment in early hematopoietic precursors.
Maurillo
L
L
Cattedra di Ematologia, Università Tor Vergata, Divisione di Ematologia, Ospedale S. Eugenio, piazzale Umanesimo 10, 00144 Rome, Italy. lucamaurillo@tiscalinet.it
Del Poeta
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G
Venditti
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Buccisano
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F
Battaglia
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Santinelli
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Caravita
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Epiceno
A M
AM
Del Moro
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B
Tamburini
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A
Picardi
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Suppo
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Catalano
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Bruno
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Amadori
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D000328:Adult; D018952:Antigens, CD34 / Q000032:analysis; D001773:Blood Cells / Q000276:immunology / Q000378:metabolism; D001854:Bone Marrow Cells / Q000276:immunology / Q000378:metabolism; D005312:Fetal Blood / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D006412:Hematopoietic Stem Cells / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D007945:Leukemia, Lymphoid / Q000473:pathology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis* / Q000378:metabolism; D019014:fas Receptor / Q000096:biosynthesis* / Q000378:metabolism
D018952:Antigens, CD34; D019253:Proto-Oncogene Proteins c-bcl-2; D019014:fas Receptor